Arsenic-induced dose-dependent modulation of the NF-κB/IL-6 axis in thymocytes triggers differential immune responses

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Choudhury, Sreetama; Gupta, Payal; Ghosh, Sayan; Mukherjee, Sudeshna; Chakraborty, Priyanka; Chatterji, Urmi; Chattopadhyay, Sreya

2016-01-01

Highlights: • We for the first time explicitly show that arsenic exposure causes morphological damage to the thymus and results in heightened death of thymocytes. • Our data suggests that arsenic-induced apoptosis occurs due to increase in cellular oxidative and nitrosative stress. • We have for the first time established a non-classical role of NF-κB, correlating it with increase in FoxP3 expression. • The % of CD4+ CD25+ T cells were high and expression of FoxP3 has also increased at higher doses of arsenic indicating an nTreg bias. - Abstract: Arsenic contamination of drinking water is a matter of global concern. Arsenic intake impairs immune responses and leads to a variety of pathological conditions including cancer. In order to understand the intricate tuning of immune responses elicited by chronic exposure to arsenic, a mouse model was established by subjecting mice to different environmentally relevant concentrations of arsenic in drinking water for 30 days. Detailed study of the thymus, a primary immune organ, revealed arsenic-mediated tissue damage in both histological specimens and scanning electron micrographs. Analysis of molecular markers of apoptosis by Western blot revealed a dose-dependent activation of the apoptotic cascade. Enzymatic assays supported oxidative stress as an instigator of cell death. Interestingly, assessment of inflammatory responses revealed disparity in the NF-κB/IL-6/STAT3 axis, where it was found that in animals consuming higher amounts of arsenic NF-κB activation did not lead to the classical IL-6 upregulation response. This deviation from the canonical pathway was accompanied with a significant rise in numbers of CD4+ CD25+ FoxP3 expressing cells in the thymus. The cytokine profile of the animals exposed to higher doses of arsenic also indicated an immune-suppressed milieu, thus validating that arsenic shapes the immune environment in context to its dose of exposure and that at higher doses it leads to immune

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

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Panich, Tipattaraporn; Tragoolpua, Khajornsak; Pata, Supansa; Tayapiwatana, Chatchai; Intasai, Nutjeera

2017-02-01

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

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Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

2015-07-01

To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzymatic activity of fungi isolated from crops

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Wioletta A. Żukiewicz-Sobczak

2016-12-01

Full Text Available Aim: To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye. Isolated strains were also classified in the scale of biosafety levels (BSL. Material and methods: The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil collected in the Lublin region (eastern Poland. API ZYM test (bioMérieux was carried out according to the manufacturer’s instructions. Classification of BSL (Biosafety levels was based on the current literature. Results : High enzymatic activity was found in strains cultured in media containing 1% of wheat grain ( Bipolaris holmi, Penicillium decumbens and with an addition of 1% of rye grain ( Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata . The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used. Conclusions : Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes. Key words: enzymatic activity, mold fungi, zymogram, biohazards.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

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Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

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Dolinska, Monika B; Kus, Nicole J; Farney, S Katie; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

2017-01-01

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Enzymatic activity of the cellulolytic complex produced by trichoderma reesei. Enzymatic hydrolysis of cellulose

International Nuclear Information System (INIS)

Alfonsel Jaen, M.; Negro, M.J.; Saez, R.; Martin Moreno, C.

1986-01-01

The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reese QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass from Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars productions, have been selected. Previous studies on enzymatic hydrolysis of O. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (author). 10 figs.; 10 refs

Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose

International Nuclear Information System (INIS)

Alfonsel J, M.; Negro A, M. J.; Saez A, R.; Martin M, C.

1986-01-01

The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs

First results on enzymatic activities in two salt marsh soils under different hydromorphic level and vegetation

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Carmen Trasar-Cepeda

2015-12-01

Full Text Available Salt-marsh soils are soils characterized by non-permanent hydric saturation that, depending on factors like duration of submersion periods, are dominated by different salt-tolerant plant species. The composition of microbial communities is an essential component in trophic dynamics and biogeochemical processes in salt marshes, and determines the level of enzymatic activities, which catalyze the conversion of complex molecules into simpler ones. Despite of this, the enzymatic activities in marsh-soils has not yet been investigated. The aim of this study was to analyze the enzymatic activities in two soil profiles of marsh-soils under different water saturation level and dominated by different plant species [Juncus maritimus Lam and Spartina maritima (Curtis Fernald (Sp]. In both soils, the enzymatic activities were much lower than the levels typically found in terrestrial ecosystems. The enzymatic activities were measured both in air-dried and in re-moistened and incubated soil samples. In air-dried samples, the enzymatic activities were higher in Juncus than in Spartina soil and tended to decrease with depth, being sharper the decrease in Juncus than in Spartina soil. Re-moistened and pre-incubated soils showed a general increase in all the enzymatic activities and throughout the whole soil profile, especially in Spartina soils. Hydrolase activities showed a strong and positive relationship with organic matter content both in air-dried and in re-moistened soil samples, higher in these latter. In general, oxidoreductase activities only showed this relationship in re-moistened soil samples. More studies, preferably using freshly collected soil samples, are needed to understand the relationship between enzymatic activities and these environmental conditions.

Impact of herbaceous vegetation on the enzymatic activity of coal mining wastes

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Osmanczyk, D

1980-01-01

Differences in the enzymatic activity of reclaimed and crude dump wastes after coal mining were investigated. Due to the increased activity of six investigated enzymes (dehydrogenase, catalase, saccharase, BETA-glucosidase, urease and asparaginase), a favourable impact of herbaceous vegetation on the biological activation of the breeding-ground was noticed. Particularly in the case of sacharase and BETA-glucosidase, an increase of the enzymatic activity at a rate of several times or even more than ten times speaks not only for an adequate increase of the metabolic rate of carbohydrates but also for specific properties of the habitat which favours an adsorption of these enzymes. (6 refs.) (In Polish)

Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

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Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

2018-01-01

Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

The human TRPV6 channel protein is associated with cyclophilin B in human placenta.

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Stumpf, Tobias; Zhang, Qi; Hirnet, Daniela; Lewandrowski, Urs; Sickmann, Albert; Wissenbach, Ulrich; Dörr, Janka; Lohr, Christian; Deitmer, Joachim W; Fecher-Trost, Claudia

2008-06-27

Transcellular calcium transport in the kidney, pancreas, small intestine, and placenta is partly mediated by transient receptor potential (TRP) channels. The highly selective TRPV6 calcium channel protein is most likely important for the calcium transfer in different specialized epithelial cells. In the human placenta the protein is expressed in trophoblast tissue, where it is implicated in the transepithelial calcium transfer from mother to the fetus. We enriched the TRPV6 channel protein endogenously expressed in placenta together with annexin A2 and cyclophilin B (CypB), which is a member of the huge immunophilin family. In the human placenta TRPV6 and CypB are mainly located intracellularly in the syncytiotrophoblast layer, but a small amount of the mature glycosylated TRPV6 channel protein and CypB is also expressed in microvilli apical membranes, the fetomaternal barrier. To understand the role of CypB on the TRPV6 channel function, we evaluated the effect of CypB co-expression on TRPV6-mediated calcium uptake into Xenopus laevis oocytes expressing TRPV6. A significant increase of TRPV6-mediated calcium uptake was observed after CypB/TRPV6 co-expression. This stimulatory effect of CypB was reversed by the immunosuppressive drug cyclosporin A, which inhibits the enzymatic activity of CypB. Cyclosporin A had no significant effect on TRPV6 and CypB protein expression levels in the oocytes. In summary, our results establish CypB as a new TRPV6 accessory protein with potential involvement in TRPV6 channel activation through its peptidyl-prolyl cis/trans isomerase activity.

Visualizing molecular juggling within a B[subscript 12]-dependent methyltransferase complex

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Kung, Yan; Ando, Nozomi; Doukov, Tzanko I.; Blasiak, Leah C.; Bender, Güne; #351; ; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L. (MIT); (Michigan); (UNL)

2013-04-08

Derivatives of vitamin B{sub 12} are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO{sub 2} fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B{sub 12} cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B{sub 12}-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B{sub 12}-dependent methyl transfer. In addition, the structures capture B{sub 12} at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B{sub 12} movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

Boron-purity-dependent Raman spectra of CaB6

International Nuclear Information System (INIS)

Song, M.; Yang, I. S.; Kim, J. Y.; Cho, B. K.

2006-01-01

We report significant differences in the Raman spectra of two different kinds of CaB 6 single crystals grown from boron with a purity of 99.9 % (3N) or 99.9999 % (6N). Our Raman spectra of CaB 6 (3N) show peaks around 781 (T 2g ), 1141 (E g ), and 1283 cm -1 (A 1g ), and they are very similar to previous Raman spectra of CaB 6 . The E g mode shows a characteristic double-peak feature due to an additional weak broad peak centered around 1158 cm -1 . However, the Raman spectra of CaB 6 (6N) show sharp peaks around 771 (T 2g ), 1137 (E g ), and 1266 cm -1 (A 1g ). The peak frequencies are down shifted as much as ∼17 cm -1 . In addition, no additional peak feature is observed for the E g mode so that the mode is symmetric in the case of CaB 6 (6N). The X-ray powder diffraction patterns for both CaB 6 (3N) and CaB 6 (6N) show that the lattice parameters are essentially the same. The majority of the impurity in the 99.9 %-pure (3N) boron is C. Thus, we doped CaB 6 (6N) with C, making Ca(B 0.995 C 0.005 ) 6 , and looked for differences in the Raman spectra. The Raman spectra of Ca(B 0.995 C 0.005 ) 6 are nearly identical to those of CaB 6 (6N), indicating that the differences between the Raman spectra of CaB 6 (3N) and CaB 6 (6N) are not due to a C impurity. The Raman results show that the presence of impurities, not the amount of them, is enough to trigger local symmetry breaking in CaB 6 . The broadening of T 2g , the additional E g2 mode and the asymmetry of A 1g in CaB 6 (3N) can be understood in terms of the symmetry of the arrangements of the boron octahedra lowered by local symmetry breaking.

Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

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Yan Huang

Full Text Available Glutamate decarboxylase (GAD catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA. In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C. Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C, superior thermostability (2.8-fold greater than that of GAD-C, and higher kcat/Km (1.6-fold higher than that of GAD-C. Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

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Hu, Gui-Qiu; Song, Pei-Xuan; Chen, Wei; Qi, Shuai; Yu, Shui-Xing; Du, Chong-Tao; Deng, Xu-Ming; Ouyang, Hong-Sheng; Yang, Yong-Jun

2017-10-01

Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected. We found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB. Copyright © 2017. Published by Elsevier Ltd.

Haplotypes frequencies of CYP2B6 in Malaysia

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N Musa

2012-01-01

Full Text Available Background: Drugs with complex pharmacology are used in the management of drug use disorder (DUD and HIV/AIDS in Malaysia and in parts of South-East Asia. Their multiethnic populations suggest complexity due to the genetic polymorphism, such as CYP2B6 that metabolizes methadone and anti-retroviral. Aims: Our aim was to explore the genetic polymorphism of CYP2B6 among Malays, Chinese, Indians, and opiate-dependent individuals in Malaysia. Settings and Design: The study utilized DNA from our previous studies on CYPs and new recruitments from opiate-dependent individuals. Materials and Methods: For the new recruitment, after obtaining consent and baseline demography, 5 ml blood was obtained from patients attending methadone maintenance therapy (MMT Clinics. Genomic DNA was extracted using standard methods. 10 nucleotide changes associated with CYP2B6FNx0110, CYP2B6FNx012, CYP2B6FNx0117, CYP2B6FNx0111, CYP2B6FNx018, CYP2B6FNx0114, CYP2B6FNx019, CYP2B6FNx014, CYP2B6FNx016, CYP2B6FNx0127, and CYP2B6FNx0120 were determined using multiplex nested allele-specific PCR. Statistical Analysis: Descriptive statistics were used to summarize demographic data. Differences in allele frequencies between populations were tested using Chi-squared test and were corrected using the Bonferroni test. Results: CYP2B6 polymorphism in Malaysia is variable with trends that suggest an ethnic difference. Reduced activity CYP2B6FNx016 occurred in 13% to 26% among Malays, Chinese, Indians and opiate-dependent individuals. Another ′reduced activity′, CYP2B6FNx012 allele, was found at much lower percentages in the groups. Conclusions: The relative commonness of reduced-activity CYP2B6 alleles in our study called for attention in terms of dosage requirements for MMT and ARV in Malaysia. It also implored follow-up association studies to determine its relevance and consequences in personalized medicine for drug use disorder and HIV/AIDS.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

International Nuclear Information System (INIS)

Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

2010-01-01

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 μM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 μM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 μM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 μM and 10 μM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

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Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

2010-06-15

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.

Development of positron tracer for in vivo estimation of brain MAO-B activity

International Nuclear Information System (INIS)

Inoue, Osamu; Tominaga, Toshiyoshi; Fukuda, Nobuo; Suzuki, Kazutoshi; Yamasaki, Toshio

1984-01-01

Both the specificity and the measurable range of enzyme activity of this method were found to be much dependent upon the enzymatic properties of substrate-tracer. The measurable range of brain enzyme activity was found to be from zero to the maximum value which was dependent upon two factors; the elimination rate of substratetracer from the brain (Ksub(el)) and the Vsub(max)/Ksub(m) value of substrate. The detectable range of changes in enzyme activity can be made wider by using another substrate as a tracer which has a lower Vsub(max)/Ksub(m) value or larger Ksub(el) value. The specificity can be also favorably designed by selection of substrate with various enzymatic or physico-chemical properties as a tracer. N, N-dimethyl phenylethylamine (DMPEA) was selected as a substrate-tracer for the estimation of brain MAO-B activity. Very high accumulation of radioactivity into mouse brain at 1 min after intravenous injection of 11 C-DMPEA, and a long-term retention of radioactivity in the brain were observed. 11 C-DMPEA seemed to be metabolized to 11 C-dimethylamine by brain MAO, and be trapped by the blood-brain barrier. When various dosage of 1-deprenyl (a specific MAO-B inhibitor) were pretreated, brain radioactivity at 1 hr after injection of 11 C-DMPEA significantly decreased in a dosage (1-deprenyl)-dependent way, while pretreatment with clorgyline (a specific MAO-A inhibitor) had no effect. This decrease in radioactivity might be due to the decrease of the production rate of labeled metabolite ( 11 C-dimethylamine) in the brain. The relationship between the radioactivity remaining at 1 hr after injection and MAO-B activity remaining in the brain was quite paralle. 11 C-DMPEA seems to be a specific radiotracer for the external detection of alterations in MAO-B activity in the brain with a fair sensitivity. (J.P.N.)

The herpes simplex virus 1-encoded envelope glycoprotein B activates NF-κB through the Toll-like receptor 2 and MyD88/TRAF6-dependent signaling pathway.

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Mingsheng Cai

Full Text Available The innate immune response plays a critical role in the host defense against invading pathogens, and TLR2, a member of the Toll-like receptor (TLR family, has been implicated in the immune response and initiation of inflammatory cytokine secretion against several human viruses. Previous studies have demonstrated that infectious and ultraviolet-inactivated herpes simplex virus 1 (HSV-1 virions lead to the activation of nuclear factor kappa B (NF-κB and secretion of proinflammatory cytokines via TLR2. However, except for the envelope glycoprotein gH and gL, whether there are other determinants of HSV-1 responsible for TLR2 mediated biological effects is not known yet. Here, we demonstrated that the HSV-1-encoded envelope glycoprotein gB displays as molecular target recognized by TLR2. gB coimmunoprecipitated with TLR2, TLR1 and TLR6 in transfected and infected human embryonic kidney (HEK 293T cells. Treatment of TLR2-transfected HEK293T (HEK293T-TLR2 cells with purified gB results in the activation of NF-κB reporter, and this activation requires the recruitment of the adaptor molecules myeloid differentiation primary-response protein 88 (MyD88 and tumor necrosis factor receptor-associated factor 6 (TRAF6 but not CD14. Furthermore, activation of NF-κB was abrogated by anti-gB and anti-TLR2 blocking antibodies. In addition, the expression of interleukin-8 induced by gB was abrogated by the treatment of the human monocytic cell line THP-1 with anti-TLR2 blocking antibody or by the incubation of gB with anti-gB antibody. Taken together, these results indicate the importance and potency of HSV-1 gB as one of pathogen-associated molecular patterns (PAMPs molecule recognized by TLR2 with immediate kinetics.

Effect of aflatoxin B1 on growth and enzymatic activity of a native strain of Bacillus sp Efecto de la aflatoxina B1 sobre el crecimiento y actividad proteolítica de una cepa nativa de Bacillus sp

Directory of Open Access Journals (Sweden)

Márquez Edna Judith

2004-07-01

Full Text Available The effect of different aflatoxin B1 (AFAB1 concentrations on alkaline protease growth and enzymatic activity was evaluated; a native strain of alkalophilic Bacillus sp cultivated in CSL (Corn Steep Liquor was used. It was found that the effect of AFAB1 on the strain inhibited its growth and enzymatic activity to 1 ppm, showing that the strain is highly sensible to AFAB1, meaning that medium obtained f rom Colombian corn contaminated with this mycotoxin cannot be easily used. Concentrations less than 0.1 ppm did not affect growth and enzymatic activity. Key words: Bacillus, aflatoxin, alkaline proteases.Se evaluó el efecto de diferentes concentraciones de aflatoxina B1 (AFAB1 sobre el crecimiento y actividad enzimática de proteasas alcalinas de una cepa nativa de Bacillus sp Alcalofílico cultivada en LAM (Licor Agotado de Maíz. Se encontró que la cepa inhibe su crecimiento y actividad enzimática a 1 ppm, lo que demuestra una alta sensibilidad de la cepa evaluada a la AFAB1 e imposibilita utilizar fácilmente medios obtenidos de maíz nacional contaminado con esta micotoxina. Las concentraciones inferiores a 0.1 ppm no tienen ningún efecto sobre el crecimiento y la actividad enzimática. Palabras clave: Bacillus, aflatoxina, proteasas alcalinas.

Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

Directory of Open Access Journals (Sweden)

Nigou Jérôme

2007-08-01

Full Text Available Abstract Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase. Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.

The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

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Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

2013-08-01

The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association

Assessment of intake and nutritional status of vitamin b1, b2, and b6 in men and women with different physical activity levels.

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Malara, M; Hübner-Wozniak, E; Lewandowska, I

2013-06-01

The purpose of the present study was to examine the nutritional status of vitamin B1, B2, and B6 in respect to dietary intake of these vitamins and activity coefficients of the erythrocyte enzymes transketolase, glutathione reductase, and aspartic aminotransferase in young men and women with different physical activity levels. The participants of this study were 20 women and 20 men with high physical activity (groups HAW and HAM, respectively), and 20 women and 20 men with low physical activity (groups LAW and LAM, respectively). The intake of vitamins B1, B2, B6, proteins, and calorie content of the diet was based on the average of the 4-day dietary recalls. To assess nutritional status of vitamin B1, B2, and B6, the activity coefficients (α) of erythrocyte transketolase (ETK), erythrocyte glutathione reductase (EGR), and erythrocyte aspartic aminotransferase (EAST) were estimated in blood hemolysates. The intake of the studied vitamins in the diet was statistically significantly lower in the female groups compared with the respective male groups. Deficiency of vitamin B6 in the diet was present more often in women than in men (in terms of the recommended dietary allowances [RDA]). Values of the activity coefficient αETK indicated that none of the groups in this study suffered the risk of vitamin B1 deficiency. The value of the activity coefficient αEGR indicated that the groups of women and men with low physical activity were more prone to vitamin B2 deficiency compared with the high physical activity groups. The risk of vitamin B6 deficiency (αEAST) in both male groups was higher than in both female groups. The obtained results do not allow for unequivocal determination of the impact of sex and the level of physical activity on intake and nutritional status of vitamin B1, B2, and B6. Independently of sex and the level of physical activity, the women and men consumed insufficient quantities of vitamins B1 and B6, although this was not always related to

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

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Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

2010-12-01

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

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Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

2005-04-01

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

Measure of enzymatic activity coincident with 2450 MHz microwave exposure

Energy Technology Data Exchange (ETDEWEB)

Ward, T R; Allis, J W; Elder, J A

1975-09-01

Enzyme preparations were exposed to microwave radiation at 2450 MHz and enzymatic activity was simultaneously monitored spectrophotometrically with a crossed-beam exposure detection system. Enzymes studied were glucose 6-phosphate dehydrogenase from human red blood cells and yeast, adenylate kinase from rat liver mitochondria and rabbit muscle, and rat liver microsomal NADPH cytochrome c reductase. No difference was found between the specific activity at 25/sup 0/C of unirradiated controls and enzyme preparations irradiated at an absorbed dose rate of 42 W/kg.

Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

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Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

2017-02-01

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.

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Elgueta, Raul; Marks, Ellen; Nowak, Elizabeth; Menezes, Shinelle; Benson, Micah; Raman, Vanitha S; Ortiz, Carla; O'Connell, Samuel; Hess, Henry; Lord, Graham M; Noelle, Randolph

2015-01-15

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (B(mem)) are essential for the secondary humoral immune responses, the chemokine response patterns of B(mem) cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific B(mem) cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of B(mem), CCR6 is essential for the ability of B(mem) to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient B(mem) was revealed by immunohistological analysis with an altered distribution of CCR6-deficient B(mem) from the marginal and perifollicular to the follicular/germinal center area. Copyright © 2015 by The American Association of Immunologists, Inc.

The role of chloride in the mechanism of O(2) activation at the mononuclear nonheme Fe(II) center of the halogenase HctB.

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Pratter, Sarah M; Light, Kenneth M; Solomon, Edward I; Straganz, Grit D

2014-07-02

Mononuclear nonheme Fe(II) (MNH) and α-ketoglutarate (α-KG) dependent halogenases activate O2 to perform oxidative halogenations of activated and nonactivated carbon centers. While the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. Here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative transformation of the Fe(II)-α-KG-enzyme complex is >200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. By contrast, the presence of substrate, which generally brings about O2 activation at enzymatic MNH centers, only has an ∼10-fold effect in the absence of chloride. Circular dichroism (CD) and magnetic CD (MCD) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable-temperature, variable-field (VTVH) MCD studies of non-α-KG-containing forms and the conversion from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound. In the presence of substrate, a site with square pyramidal five-coordinate (5C) geometry is observed, which is required for O2 activation at enzymatic MNH centers. In the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. Molecular dynamics simulations suggest that the binding of chloride to the metal center of HctB leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme-substrate complex. Results are discussed in relation to other MNH dependent halogenases.

Detection of extracellular enzymatic activity in microorganisms ...

African Journals Online (AJOL)

Detection of extracellular enzymatic activity in microorganisms isolated from waste vegetable oil contaminated soil using plate methodologies. Eugenia G. Ortiz Lechuga, Isela Quintero Zapata, Katiushka Arévalo Niño ...

Detection of extracellular enzymatic activity in microorganisms ...

African Journals Online (AJOL)

sunny t

2015-09-18

Sep 18, 2015 ... microorganisms with all three enzymatic activities, thereby establishing these techniques as ... supplemented at 1% with vegetable oils, including olive (OLI) ..... cepacia lipase for biodiesel fuel production from soybean oil.

Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

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Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

2015-04-01

On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

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Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

2015-04-01

Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s). © 2015 FEBS.

Biosensing strategies based on enzymatic reactions and nanoparticles.

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Díez-Buitrago, Beatriz; Briz, Nerea; Liz-Marzán, Luis M; Pavlov, Valeri

2018-04-16

Enzymes are pivotal elements in bioanalysis due to their specificity and extremely high catalytic activity. The sensitivity of bioanalytical assays depends mainly on the capacity of an observer to detect the product(s) of a biocatalytic reaction. Both natural and artificial compounds have been traditionally used to evaluate enzymatic activities. The drawbacks of chromogenic and fluorogenic organic enzymatic substrates are their high cost and low stability, resulting in high background signals. We review here state of the art assays in the detection of enzymatic activities using recent advances in nanoscience. Novel methods based on the use of nanoparticles lead to increased sensitivity and decreased costs for bioanalysis based on enzymes as recognition elements and signal amplifiers in Enzyme-Linked Immunosorbent Assays (ELISA). Novel approaches toward the detection of enzymatic activities are based on biocatalytic synthesis, modulation, etching, and aggregation of nanoparticles under physiological conditions.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

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Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

2015-06-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

Enzymatic Hydrolysis of Oleuropein from Olea europea (Olive Leaf Extract and Antioxidant Activities

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Jiao-Jiao Yuan

2015-02-01

Full Text Available Oleuropein (OE, the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

Science.gov (United States)

Luangwattananun, Piriya; Eiamphungporn, Warawan; Songtawee, Napat; Bülow, Leif; Isarankura Na Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Yainoy, Sakda

2017-04-10

Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Confirmation of mutations in PROSC as a novel cause of vitamin B 6 -dependent epilepsy.

Science.gov (United States)

Plecko, Barbara; Zweier, Markus; Begemann, Anaïs; Mathis, Deborah; Schmitt, Bernhard; Striano, Pasquale; Baethmann, Martina; Vari, Maria Stella; Beccaria, Francesca; Zara, Federico; Crowther, Lisa M; Joset, Pascal; Sticht, Heinrich; Papuc, Sorina Mihaela; Rauch, Anita

2017-12-01

Vitamin-B 6 -dependent epilepsies are a heterogenous group of treatable disorders due to mutations in several genes ( ALDH7A1, PNPO, ALPL or ALDH4A1 ). In neonatal seizures, defects in ALDH7A1 and PNPO explain a major fraction of cases. Very recently biallelic mutations in PROSC were shown to be a novel cause in five families. We identified four further unrelated patients harbouring a total of six different mutations, including four novel disease mutations. Vitamin B 6 plasma profiles on pyridoxine did not enable the differentiation of patients with PROSC mutations. All four patients were normocephalic and had normal cranial imaging. Pyridoxine monotherapy allowed complete seizure control in one, while two patients had occasional febrile or afebrile seizures and one needed additional valproate therapy for photosensitive seizures. Two patients underwent a controlled pyridoxine withdrawal with signs of encephalopathy within a couple of days. Three had favourable outcome with normal intellectual properties at age 12.5, 15.5 and 30 years, respectively, while one child had marked developmental delay at age 27 months. The clinical and electroencephalographic phenotype in patients with PROSC mutations was indistinguishable from ALDH7A1 and PNPO deficiency. We therefore confirm PROSC as a novel gene for vitamin-B 6 -dependent epilepsy and delineate a non-specific plasma vitamin B 6 profile under pyridoxine treatment. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Heavy metal pollution and soil enzymatic activity

Energy Technology Data Exchange (ETDEWEB)

Tyler, G

1974-01-01

The activity of hydrolytic soil enzymes was studied on spruce mor, polluted with Cu and Zn from a brass foundry in Sweden. Approximately straight regression lines were obtained between enzymatic activity or respiration rate and log Cu + Zn concentration, with highly significant negative regression coefficients for urease and acid phosphatase activity as well as respiration rate, whereas US -glucosidase activity was not measurably lower at high concentrations of Cu + Zn. 17 references, 5 figures.

Variations in Enzymatic Activities of Shoots and Roots as an Indicator for Irradiated Seeds

International Nuclear Information System (INIS)

Abdelbbaary, N.A.; Elagamay, M.R.

2005-01-01

Germinated seedlings from oil seeds (sesame and sunflower) and legumes (Trigonella, Haricot, broad bean and cow pea) were irradiated with gamma rays at doses of 0, 0.2, 0.4, 0.8 and 1 kGy and the data were collected from shoots and roots. Enzymatic activities appeared to be correlated with gamma irradiation dose. The enzymatic activities of irradiated seeds understudy were significantly higher than controls. The peroxidase activities were nearly similar in both roots and shoots, while acid phosphatase activities in roots were higher than in shoots. Also protein contents were higher in roots. The peroxidase and acid phosphatase specific activities in roots were similar. Shoots peroxidase enzymatic activity increased with increased gamma doses. The seedling under study showed two different levels of peroxidase activity, higher as sesame, Trigonella and Sunflower, and lower such as all other legumes understudy. Similar tendency have been also noticed in roots-enzymatic activity, positive correlation between gamma doses treatment and peroxidase enzymatic activity, again two groups higher activity cow pea, broad bean, bean and Trigonella lower such as sesame, such as sesame, sunflower and haircut

Activation of NF-κB is involved in 6-hydroxydopamine-but not MPP+-induced dopaminergic neuronal cell death: its potential role as a survival determinant

International Nuclear Information System (INIS)

Park, Seong H.; Choi, Won-Seok; Yoon, So-Young; Ahn, Young Soo; Oh, Young J.

2004-01-01

The nuclear factor-kappaB (NF-κB) family plays an important role in the control of the apoptotic response. Its activation has been demonstrated in both neurons and glial cells in many neurological disorders. In the present study, we specifically examined whether and to what extent NF-κB activation is involved in culture models of Parkinson's disease following exposure of MN9D dopaminergic neuronal cells to 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-4-phenylpyridinium ion (MPP + ). Both analysis by immunocytochemistry and of immunoblots revealed that NF-κB-p65 was translocated into the nuclei following 6-OHDA but not MPP + -treatment. A time-dependent activation of NF-κB induced by 6-OHDA but not MPP + was also demonstrated by an electrophoretic mobility shift assay. A competition assay indicated that not only NF-κB-p65 but also -p50 is involved in 6-OHDA-induced NF-κB activity. Co-treatment with an antioxidant, N-acetyl-L-cysteine, blocked 6-OHDA-induced activation of NF-κB signaling. In the presence of an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), 6-OHDA-induced cell death was accelerated while PDTC did not affect MPP + -induced cell death. Our data may point to a drug-specific activation of NF-κB as a survival determinant for dopaminergic neurons

Gamma radiation effect on biological activity and enzymatic properties of snake venoms

International Nuclear Information System (INIS)

Herrera, E.; Yarleque, A.; Campos, S.; Zavaleta, A.

1986-01-01

The effect of gamma radiation, from Co-60, on the biological activity and on some enzymatic activities, present in the venoms of Lachesis muta and Bothrops atrox, using samples of dried venom that had been irradiated at a dose of 0.1, 0.5 and 1.0 Mrad have been studied. Variations in the degree of hemorrhage and local necrosis were observed in albino mice injected subcutaneously with venoms of both types. The reduction of the biological activity was greater for the local hemorrhagic effect and was dependent on the doses of irradiation. The specific activity of various enzymes, present in both venoms, is affected by the gamma radiation, at a dose of 0.1 Mrad the order of increasing inactivation being: exonuclease (4%), phospholipase (24%), caseinolytic enzyme (20%), tamesterase (33%), a thrombine-like enzyme (40%), fibrinolytic enzyme (41%), 5'-nucleotidase (50%) and endonuclease (55%). The enzymatic inactivation was augmented by 0.5 and 1.0 Mrad, without maintaining an arithmetic relation. The enzyme of major resistance to the radiation was exonuclease, whereas 5'-nucleotidase and endonuclease were the most sensitive. No significant changes were observed in the spectrum of UV absorbtion (range 260 to 290 nm) nor in the contents of L-tyrosine in the irradiated venoms

Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

International Nuclear Information System (INIS)

Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

2013-01-01

Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation

Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

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Lv, Peng [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Xue, Peng; Dong, Jian [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Peng, Hui [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Clewell, Rebecca [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Wang, Aiping [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Wang, Yue [Institute for Medical Device Standardization Administration, National Institutes for Food and Drug Control, Beijing (China); Peng, Shuangqing [Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Qu, Weidong [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Zhang, Qiang; Andersen, Melvin E. [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States)

2013-11-01

Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

Atividade da 6-fosfogliconato desidrogenase em deficientes de glicose-6-fosfato desidrogenase Activity of 6-phosphogluconate dehydrogenase in glucose-6-phosphate dehydrogenase deficiency

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Daniela B. Nicolielo

2006-06-01

to know better the actuation of these enzymes. The goal of this study was to evaluate the 6PGD enzymatic activity on a population with G6PD deficiency, to verify if there is an elevation of the activity of this enzyme, and try to correlate to a possible increase on the number of reticulocytes or the presence of alterations on red series. The research with 2657 male individuals detected 97 deficient for G6PD, which determined a 3.65% prevalence for the Bauru (SP region, with mean enzymatic activity of 1.74 UI.g Hb-1. min-1 at 37ºC, 14,4% of the normal G6PD activity. Mean 6PGD enzymatic activity was 9.5 UI.g Hb-1. min-1 at 37ºC, and was elevated in 47.4% of the G6PD deficient individuals. The result obtained did not confirm the hypothesis that the elevation of the 6PGD enzymatic activity, in G6PD deficient individuals, was due to the presence of an increase of reticulocytes in blood stream, age or erythrocytometric alterations that could denote anemia. The most plausible theory is that the auto-limited hemolysis, imposed by oxidative processes, preserves young erythrocytes that have an elevated enzymatic activity, as naturally these enzymes lose activity with cellular aging.

DNA-dependent protein kinase participates in the radiation activation of NF-kB

International Nuclear Information System (INIS)

Rosenzweig, Kenneth E.; Youmell, Matthew B.; Price, Brendan D.

1997-01-01

The NF-kB transcription factor is maintained in an inactive state by binding to the lkBa inhibitory protein. Activation requires phosphorylation and degradation of lkBa, releasing active NF-kB. NF-kB can be activated by cytokines, antigens, free radicals and X-ray irradiation. The protein kinase responsible for phosphorylation of lkBa in vivo has not been fully characterized. Here, we have examined the role of the DNA-dependent protein kinases (DNA-PK) in the radiation-activation of NF-kB. Wortmannin is an inhibitor of DNA-PK and related kinases. Exposure of SW480 cells to wortmannin inhibited the radioactivation of NF-kB DNA-binding. Analysis of lkBa levels by western blotting indicated that wortmannin blocked the radiation induced degradation of lkBa. In in vitro experiments, purified DNA-PK was able to efficiently phosphorylate lkBa, and this phosphorylation was inhibited by wortmannin. In contrast, the induction of NF-kB activity by TNFa was unaffected by wortmannin. The results suggest that DNA-PK may phosphorylate lkBa following irradiation, leading to degradation of lkBa and the release of active NF-kB. The inability of wortmannin to block TNFa activation of NF-kB indicates there may be more than one pathway for the activation of NF-kB

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

Science.gov (United States)

Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

2014-04-01

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

Science.gov (United States)

Nakaya, K; Mizuno, R; Ohhashi, T

2001-12-01

We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

Enzymatic activity measured by microcalorimetry in soil amended with organic residues

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Karina Cenciani

2011-08-01

Full Text Available Enzymatic activity is an important property for soil quality evaluation. Two sequences of experiments were carried out in order to evaluate the enzymatic activity in a soil (Rhodic Eutrudox amended with cattle manure, earthworm casts, or sewage sludges from the municipalities of Barueri and Franca. The activity of commercial enzymes was measured by microcalorimetry in the same soil samples after sterilization. In the first experiment, the enzyme activities of cellulase, protease, and urease were determined in the soil samples during a three month period. In the second sequence of experiments, the thermal effect of the commercial enzymes cellulase, protease, and urease on sterilized soil samples under the same tretaments was monitored for a period of 46 days. The experimental design was randomized and arranged as factorial scheme in five treatments x seven samplings with five replications. The treatment effects were statistically evaluated by one-way analysis of variance. Tukey´s test was used to compare means at p < 0.05. The presence of different sources of organic residues increased the enzymatic activity in the sampling period. Cattle manure induced the highest enzymatic activity, followed by municipal sewage sludge, whereas earthworm casts induced the lowest activity, but differed from control treatment. The thermal effect on the enzyme activity of commercial cellulase, protease, and urease showed a variety of time peaks. These values probably oscillated due to soil physical-chemical factors affecting the enzyme activity on the residues.

Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation.

Science.gov (United States)

Mathy-Hartert, M; Hogge, L; Sanchez, C; Deby-Dupont, G; Crielaard, J M; Henrotin, Y

2008-07-01

Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.

Theoretical investigation of molecular hydrogen reactions with active centres in B6H5- and AlB6H5- clusters

International Nuclear Information System (INIS)

Mebel', A.M.; Charkin, O.P.

1991-01-01

Nonempirical calculations of sections of potential surface (PS) along the shortest way of reaction of hydrogen interaction with different active centres in AlB 5 H 5 - cluster were conducted. Mechanisms of reactions of valent-saturated hydrides (BH, AlH) and clusters (B 6 H 5 - , AlB 5 H 5 - ) with molecular hydrogen are correlated. Qualitative model enabling to form an opinion about the presence or the absence of barrier on PS of the shortest way of reactions of breaking σ-bond of H-H type on the active centre of cluster, is suggested. The model is based on analysis of the character of canonical MO reagents and products

Direct electrocatalytic reduction of coenzyme NAD{sup +} to enzymatically-active 1,4-NADH employing an iridium/ruthenium-oxide electrode

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Ullah, Nehar, E-mail: nehar.ullah@mail.mcgill.ca; Ali, Irshad; Omanovic, Sasha

2015-01-15

A thermally prepared iridium/ruthenium-oxide coating (Ir{sub 0.8}Ru{sub 0.2}-oxide) formed on a titanium substrate was investigated as a possible electrode for direct electrochemical regeneration of enzymatically-active 1,4-NADH from its oxidized form NAD{sup +}, at various electrode potentials, in a batch electrochemical reactor. The coating surface was characterized by ‘cracked mud’ morphology, yielding a high surface roughness. The NADH regeneration results showed that the percentage of enzymatically-active 1,4-NADH present in the product mixture (i.e. recovery) is strongly dependent on the electrode potential, reaching a maximum (88%) at −1.70 V vs. MSE. The relatively high recovery was explained on the basis of availability of adsorbed ‘active’ hydrogen (H{sub ads}) on the Ir/Ru-oxide surface, i.e. on the basis of electrochemical hydrogenation. - Highlights: • Ir{sub 0.8}Ru{sub 0.2}-oxide coating was formed thermally on a Ti substrate. • Electrochemical regeneration of enzymatically-active 1,4-NADH was investigated. • The 1,4-NADH recovery percentage is strongly dependent on the electrode potential. • A highest recovery, 88%, was obtained at −1.70 V vs. MSE. • The NADH regeneration process involved electrochemical hydrogenation.

Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

Science.gov (United States)

Winter, Martin; Bretschneider, Tom; Kleiner, Carola; Ries, Robert; Hehn, Jörg P; Redemann, Norbert; Luippold, Andreas H; Bischoff, Daniel; Büttner, Frank H

2018-07-01

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC 50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism.

Science.gov (United States)

Valles, Soraya L; Benlloch, María; Rodriguez, María L; Mena, Salvador; Pellicer, José A; Asensi, Miguel; Obrador, Elena; Estrela, José M

2013-03-22

Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms. Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic foci using high-performance cell sorting. Stress hormones and IL-6 levels were measured by ELISA, and CRH expression in the brain by in situ hybridization. DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6 was measured using specific transcription factor assay kits. IL-6 expression was measured by RT-PCR, and silencing was achieved by transfection of anti-IL-6 small interfering RNA. GSH was determined by HPLC. Cell death analysis was distinguished using fluorescence microscopy, TUNEL labeling, and flow cytometry techniques. Statistical analyses were performed using Student's t test. Plasma levels of stress-related hormones (adrenocorticotropin hormone, corticosterone, and noradrenaline) increased, following a circadian pattern and as compared to non-tumor controls, in mice bearing B16-F10 lung or liver metastases. Corticosterone and noradrenaline, at pathophysiological levels, increased expression and secretion of IL-6 in B16-F10 cells in vitro. Corticosterone- and noradrenaline-induced transcriptional up-regulation of IL-6 gene involves changes in the DNA binding activity of nuclear factor-κB, cAMP response element-binding protein, activator protein-1, and nuclear factor for IL-6. In vivo inoculation of B16-F10 cells transfected with anti-IL-6-siRNA, treatment with a glucocorticoid receptor blocker (RU-486) or with a β-adrenoceptor blocker (propranolol), increased hepatic GSH whereas decreased plasma IL-6 levels and metastatic growth. Corticosterone, but not NORA, also induced apoptotic cell death in

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

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Tsubasa Munakata

2007-09-01

Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen

Science.gov (United States)

Elgueta, Raul; Marks, Ellen; Nowak, Elizabeth; Menezes, Shinelle; Benson, Micah; Raman, Vanitha S.; Ortiz, Carla; O’Connell, Samuel; Hess, Henry; Lord, Graham M.; Noelle, Randolph

2014-01-01

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to insure successful cognate interactions. While cognate interactions between T cells and memory B cells (Bmem)5 are essential for the secondary humoral immune responses, the chemokine response patterns of Bmem cells are largely unknown. In contrast to naïve B cells, this study shows that antigen-specific Bmem cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. While CCR6 appears be non-essential for the initial clonal expansion and maintenance of Bmem, CCR6 is essential for the ability of Bmem to respond to a recall response to their cognate antigen. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone-marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient Bmem was revealed by immunohistological analysis with an altered distribution of CCR6-deficient Bmem from the marginal and perifollicular to the follicular/germinal center area. PMID:25505290

Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil

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LAUREANA V. SOBRAL

2017-10-01

Full Text Available ABSTRACT To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%, Penicillium (21%, Talaromyces (14%, Curvularia and Paecilomyces (7% each. Aspergillus sydowii (Bainier & Sartory Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

The variations of enzymatic activity of pepsin preparation by γ-irradiation

International Nuclear Information System (INIS)

Kimura, Syojiro; Taimatsu, Meiko; Kanbashi, Toshitaka; Okamoto, Shinichi; Ohnishi, Tokuhiro.

1993-01-01

Effect of γ-irradiation on the enzymatic activity of raw pepsin and some saccharated pepsin preparations were studied in the dose range from 0 to 300 kGy. As a result, the apparent reduction rate of saccharated pepsin preparations is less than of raw pepsin. K values of raw and saccharated pepsins were 0.014 and 0.0040-0.0061, and G values of raw and saccharated pepsins were 3.98 and 1.13-1.73, respectively. The lower K and G values of saccharated pepsin than those of raw pepsin seem to be due to radiolytic products of lactose in the preparations as an excipient. Retention rates of enzymatic activity of irradiated preparations at the dose of 25 kGy, which is a complete sterilization dose of pharmaceutical materials, were estimated to be 83% for raw pepsin, and 86% and 93% for saccharated pepsin preparations. At the dose of 10 kGy suggested for food irradiation the retention rates were more than 93% for all pepsins. Therefore, this method is applicable considering the stability of the enzymatic activity after irradiation in the proper range of dose. However, it is necessary to consider the fact that radiolytic products of lactose affect the measurement of enzymatic activity. (author)

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

International Nuclear Information System (INIS)

Chen, Zhi-Dong; Xu, Liang; Tang, Kan-Kai; Gong, Fang-Xiao; Liu, Jing-Quan; Ni, Yin; Jiang, Ling-Zhi; Hong, Jun; Han, Fang; Li, Qian; Yang, Xiang-Hong; Sun, Ren-Hua; Mo, Shi-Jing

2016-01-01

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF-κB-dependent

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhi-Dong [Department of Critical Care Medicine, The First Affiliated Hospital of Huzhou Normal College, Huzhou 313000, Zhejiang (China); Xu, Liang [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Tang, Kan-Kai [Department of Critical Care Medicine, The First Affiliated Hospital of Huzhou Normal College, Huzhou 313000, Zhejiang (China); Gong, Fang-Xiao; Liu, Jing-Quan; Ni, Yin; Jiang, Ling-Zhi; Hong, Jun; Han, Fang; Li, Qian; Yang, Xiang-Hong [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Sun, Ren-Hua, E-mail: jqin168@hotmail.com [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China); Mo, Shi-Jing, E-mail: msj860307@163.com [Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, Hangzhou 310000, Zhejiang (China)

2016-09-10

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF-κB-dependent

Asymmetric total synthesis of 6-Tuliposide B and its biological activities against tulip pathogenic fungi.

Science.gov (United States)

Shigetomi, Kengo; Omoto, Shoko; Kato, Yasuo; Ubukata, Makoto

2011-01-01

The structure-activity relationship was investigated to evaluate the antifungal activities of tuliposides and tulipalins against tulip pathogenic fungi. 6-Tuliposide B was effectively synthesized via the asymmetric Baylis-Hillman reaction. Tuliposides and tulipalins showed antifungal activities against most of the strains tested at high concentrations (2.5 mM), while Botrytis tulipae was resistant to tuliposides. Tulipalin formation was involved in the antifungal activity, tulipalin A showed higher inhibitory activity than 6-tuliposide B and tulipalin B. Both the tuliposides and tulipalins showed pigment-inducing activity against Gibberella zeae and inhibitory activity against Fusarium oxysporum f. sp tulipae. These activities were induced at a much lower concentration (0.05 mM) than the antifungal MIC values.

Rapid tryptic mapping using enzymatically active mass spectrometer probe tips

Energy Technology Data Exchange (ETDEWEB)

Dogruel, D.; Williams, P.; Nelson, R.W. [Arizona State Univ., Tempe, AZ (United States)

1995-12-01

A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. 19 refs., 6 figs., 2 tabs.

IL-6 Production by TLR-Activated APC Broadly Enhances Aged Cognate CD4 Helper and B Cell Antibody Responses In Vivo.

Science.gov (United States)

Brahmakshatriya, Vinayak; Kuang, Yi; Devarajan, Priyadharshini; Xia, Jingya; Zhang, Wenliang; Vong, Allen Minh; Swain, Susan L

2017-04-01

Naive CD4 T cell responses, especially their ability to help B cell responses, become compromised with aging. We find that using APC pretreated ex vivo with TLR agonists, polyinosinic-polycytidylic acid and CpG, to prime naive CD4 T cells in vivo, restores their ability to expand and become germinal center T follicular helpers and enhances B cell IgG Ab production. Enhanced helper responses are dependent on IL-6 production by the activated APC. Aged naive CD4 T cells respond suboptimally to IL-6 compared with young cells, such that higher doses are required to induce comparable signaling. Preactivating APC overcomes this deficiency. Responses of young CD4 T cells are also enhanced by preactivating APC with similar effects but with only partial IL-6 dependency. Strikingly, introducing just the activated APC into aged mice significantly enhances otherwise compromised Ab production to inactivated influenza vaccine. These findings reveal a central role for the production of IL-6 by APC during initial cognate interactions in the generation of effective CD4 T cell help, which becomes greater with age. Without APC activation, aging CD4 T cell responses shift toward IL-6-independent Th1 and CD4 cytotoxic Th cell responses. Thus, strategies that specifically activate and provide Ag to APC could potentially enhance Ab-mediated protection in vaccine responses. Copyright © 2017 by The American Association of Immunologists, Inc.

Enzymatic browning control in cut apples (Red delicious through a system of active packaging

Directory of Open Access Journals (Sweden)

Felipe Jadán Piedra

2017-03-01

Full Text Available Enzymatic browning is one of the most relevant mechanisms of deterioration that take place in fresh-cut fruit and vegetables, as a consequence of the activity of the polyphenol oxidase enzyme on the phenolic compounds release after cellular lysis . This work is focused on the reduction of these enzymatic activity by an active packaging technology, which make use of a material that incorporates antioxidant active agents. Thus, films of ethylene-vynil alcohol copolymer (EVOH containing a typical food antioxidant, such as ascorbic acid and a polyphenol oxidase-inhibiting agent, the 4-hexylresorcinol have been developed and used to wrap apple slices. The evolution of color, the enzymatic activity and the kinetic of agents release to food simulants were monitored. The results showed an improvement of apple slice color stability and a reduction of the enzymatic activity. The film with 10 % of agents in 3/1 ratio (4-hexylresorcinol/ascorbic acid provided the best results.

Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration

Energy Technology Data Exchange (ETDEWEB)

Kang, Liang [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hu, Jia [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Weng, Yuxiong [Department of Hand Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Jia, Jie [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zhang, Yukun, E-mail: zhangyukuncom@126.com [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China)

2017-03-15

Intervertebral disc degeneration (IDD) is marked by imbalanced metabolism of the extracellular matrix (ECM) in the nucleus pulposus (NP) of intervertebral discs. This study aimed to determine whether sirtuin 6 (SIRT6), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, protects the NP from ECM degradation in IDD. Our study showed that expression of SIRT6 markedly decreased during IDD progression. Overexpression of wild-type SIRT6, but not a catalytically inactive mutant, prevented IL-1β-induced NP ECM degradation. SIRT6 depletion by RNA interference in NP cells caused ECM degradation. Moreover, SIRT6 physically interacted with nuclear factor-κB (NF-κB) catalytic subunit p65, transcriptional activity of which was significantly suppressed by SIRT6 overexpression. These results suggest that SIRT6 prevented NP ECM degradation in vitro via inhibiting NF-κB-dependent transcriptional activity and that this effect depended on its deacetylase activity. - Highlights: • SIRT6 expression is decreased in degenerative nucleus pulposus (NP) tissues. • SIRT6 overexpression lowers IL-1β-induced matrix degradation of NP. • SIRT6 inhibition induces matrix degradation of NP. • SIRT6 prevents matrix degradation of NP via the NF-κB signaling pathway.

Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration

International Nuclear Information System (INIS)

Kang, Liang; Hu, Jia; Weng, Yuxiong; Jia, Jie; Zhang, Yukun

2017-01-01

Intervertebral disc degeneration (IDD) is marked by imbalanced metabolism of the extracellular matrix (ECM) in the nucleus pulposus (NP) of intervertebral discs. This study aimed to determine whether sirtuin 6 (SIRT6), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, protects the NP from ECM degradation in IDD. Our study showed that expression of SIRT6 markedly decreased during IDD progression. Overexpression of wild-type SIRT6, but not a catalytically inactive mutant, prevented IL-1β-induced NP ECM degradation. SIRT6 depletion by RNA interference in NP cells caused ECM degradation. Moreover, SIRT6 physically interacted with nuclear factor-κB (NF-κB) catalytic subunit p65, transcriptional activity of which was significantly suppressed by SIRT6 overexpression. These results suggest that SIRT6 prevented NP ECM degradation in vitro via inhibiting NF-κB-dependent transcriptional activity and that this effect depended on its deacetylase activity. - Highlights: • SIRT6 expression is decreased in degenerative nucleus pulposus (NP) tissues. • SIRT6 overexpression lowers IL-1β-induced matrix degradation of NP. • SIRT6 inhibition induces matrix degradation of NP. • SIRT6 prevents matrix degradation of NP via the NF-κB signaling pathway.

Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

International Nuclear Information System (INIS)

Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

2003-01-01

A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6

Energy Technology Data Exchange (ETDEWEB)

Seibold, Kristina; Ehrenschwender, Martin, E-mail: martin.ehrenschwender@ukr.de

2015-08-14

CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified the multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.

PTP1B is a negative regulator of interleukin 4–induced STAT6 signaling

Science.gov (United States)

Lu, Xiaoqing; Malumbres, Raquel; Shields, Benjamin; Jiang, Xiaoyu; Sarosiek, Kristopher A.; Natkunam, Yasodha

2008-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4–induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A “substrate-trapping” mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)–inducible manner. We delineate a new negative regulatory loop of IL-4–JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase–dependent manner and enhances PTP1B protein stability to suppress IL-4–induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell–like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4–induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes. PMID:18716132

Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

Directory of Open Access Journals (Sweden)

Leandro C de Oliveira

Full Text Available Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5 was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105. In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

Effect of restricted motion in high temperature on enzymatic activity of the pancreas

Science.gov (United States)

Abdusattarov, A.; Smirnova, G. I.

1980-01-01

Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

Enzymatic Synthesis of Furfuryl Alcohol Ester with Oleic Acid by Candida antarctica Lipase B and Its Kinetic Study

Science.gov (United States)

Sengupta, Avery; Dey, Tanmoy; Ghosh, Mahua; Ghosh, Jaydip; Ghosh, Santinath

2012-08-01

This study investigated the successful enzymatic production of furfuryl oleate and its detailed kinetic study by Michaelis-Menten model. Esterification of oleic acid and furfuryl alcohol by Candida antarctica lipase B (Novozym 435 preparation) in a solvent free system was studied in the present work at 1:1 molar ratio of furfuryl alcohol and oleic acid. About 99 % conversion (on the basis of oleic acid) has been achieved within 6 h at 5 % enzyme concentration. Ping-pong bi-bi mechanism (inhibition phenomenon taken into account) was applied to describe the ratios as a complex kinetic model. The kinetic parameters were determined using MATLAB language programme. The two initial rate constants KA and KB respectively were found out by different progress curves plotted with the help of MATLAB language programme. It was concluded from the results that furfuryl alcohol considerably inhibited the enzymatic reaction while oleic acid had negligible inhibitory effect. It was clearly seen that the initial rate was increased with the increase in the furfuryl alcohol concentration until 2 M/L after which there was a drop in the initial rate depicting the inhibitory effect of furfuryl alcohol. Surprisingly, it has been observed that addition of 0.1 mol of product activated the esterification reaction. Finally, the model was found to be statistically fitting well with the experimental data.

N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

DEFF Research Database (Denmark)

Frandsen, Henrik Lauritz; Alexander, J.

2000-01-01

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N-hydroxylation followed by phase II esterification, Incubation of N...

Experimental and metabolic modeling evidence for a folate-cleaving side-activity of ketopantoate hydroxymethyltransferase (PanB

Directory of Open Access Journals (Sweden)

Jennifer eThiaville

2016-03-01

Full Text Available Tetrahydrofolate (THF and its one-carbon derivatives, collectively termed folates, are essential cofact¬ors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molec-ules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-pro¬¬ducts. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydr¬oxymethyl-7,8-dihydropterin pyrophosphokinase (FolK in many sequenced bacterial gen-omes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that over-expression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxy¬methyl-pterin and other pterins in cells and medium, and – unexpectedly – to a 46% increase in total fol-ate content. In silico modeling of the folate bio¬syn¬thesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynth-esis by folates or their damage products.

Juvenile Hormone Analogues, Methoprene and Fenoxycarb Dose-Dependently Enhance Certain Enzyme Activities in the Silkworm Bombyx Mori (L

Directory of Open Access Journals (Sweden)

M. Rajeswara Rao

2008-06-01

Full Text Available Use of Juvenile Hormone Analogues (JHA in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 μg and 3.0fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT and alanine aminotransaminase (ALAT, adenosine triphosphate synthase (ATPase and cytochrome-c-oxidase (CCO activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues.

Deacetylation of topoisomerase I is an important physiological function of E. coli CobB

Science.gov (United States)

Zhou, Qingxuan; Zhou, Yan Ning; Jin, Ding Jun

2017-01-01

Abstract Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation–deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase. PMID:28398568

Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

Science.gov (United States)

Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

2009-04-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner

DEFF Research Database (Denmark)

Kofod-Olsen, Emil; Ross-Hansen, Katrine; Mikkelsen, Jacob Giehm

2008-01-01

Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17-U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19...... transactivated the RANTES promoter. Mutational analysis of the promoter indicated that transactivation was not critically dependent on the promoter sites CRE, NF-kappaB, ISRE or NF-IL6. ND10 are nuclear substructures that are involved in several cellular regulatory pathways, including those controlling gene...... structure, U19 also localized to the centre of ND10. Knockdown of PML by small interfering RNA did not prevent U19 localization to ND10-like foci, but instead led to a fourfold increase in U19-induced transcription from the RANTES promoter. Generation of four truncated U19 proteins indicated that the N...

High field magnetoresistance and de Haas-van Alphen effect in antiferromagnetic PrB6 and NdB6

International Nuclear Information System (INIS)

Onuki, Y.; Umezawa, A.; Kwok, W.K.; Crabtree, G.W.; Nishihara, M.; Yamazaki, T.; Omi, T.; Komatsubara, T.

1987-08-01

The transport properties and the de Haas-van Alphen (dHvA) effect have been measured for antiferromagnetic PrB 6 and NdB 6 . The number of conduction electrons is approximately one per unit cell. The magnetoresistance shows the existence of open orbits implying a multiply connected Fermi surface. The angular dependence of the magnetoresistance is roughly similar to that of the reference material, LaB 6 . The dHvA data in PrB 6 shows both paramagnetic and antiferromagnetic Fermi surfaces. The antiferromagnetic Fermi surface arises from new magnetic Brillouin zone boundaries and antiferromagnetic gaps introduced by the magnetic order, and the paramagnetic Fermi surface from magnetic breakdown through the small antiferromagnetic gaps in high field. Hybridization between the conduction electrons and the f electrons has been observed through the cyclotron masses, which in PrB 6 are three times larger than the corresponding masses of LaB 6 . In NdB 6 only the antiferromagnetic Fermi surface, quite different from those of LaB 6 and PrB 6 , has been observed. 26 refs., 10 figs., 3 tabs

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B.

Science.gov (United States)

Yadav, Manish G; Kavadia, Monali R; Vadgama, Rajeshkumar N; Odaneth, Annamma A; Lali, Arvind M

2017-11-26

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2 h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.

Enzymatic activities of Azotobacter chroococcum and survival in ...

African Journals Online (AJOL)

Enzymatic activities of Azotobacter chroococcum and survival in schloropyrifos amended sterile and non-sterile. M Shukla, V Kumar, RL Thakur, N Narula. Abstract. No Abstract. Cameroon Journal of Experimental Biology Vol. 2 (2) 2006: pp. 88-94. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for ...

P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

Science.gov (United States)

Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

2007-11-01

Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and –B Enzymatic Activity

Science.gov (United States)

Brooks, Allen F.; Shao, Xia; Quesada, Carole A.; Sherman, Phillip; Scott, Peter J.H.; Kilbourn, Michael R.

2017-01-01

The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed as an in vivo marker of neuroinflammation associated with Alzheimer’s disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors, or high-affinity reversibly-binding inhibitors. As an alternative approach, we have investigated 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yields 1-[11C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brain. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain the phenyl ether demonstrated MAO-A selectivity, and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity. PMID:26393369

A role for adhesion molecules in contact-dependent T help for B cells

DEFF Research Database (Denmark)

Owens, T

1991-01-01

The role of cell contact in T-dependent B cell activation was examined. Small resting B cells from C57BL/6 mice were cultured with CBA-derived, non-alloreactive cloned T helper cells in anti-T cell receptor V beta 8-coated microwells. This induced polyclonal B cell activation to enter cell cycle...... that continued cell contact involving adhesion/accessory molecules induces B cells to proliferate and to respond to T cell lymphokines. A signaling role for cell interaction molecules on B cells is proposed, similar to the role of these and analogous molecules on T cells....

Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

Science.gov (United States)

Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

2009-08-12

The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

The enzymatic activities of brain catechol-O-methyltransferase (COMT) and methionine sulphoxide reductase are correlated in a COMT Val/Met allele-dependent fashion.

Science.gov (United States)

Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M; Hairston, Jenaqua; Bortolato, Marco

2015-12-01

The enzyme catechol-O-methyltransferase (COMT) plays a primary role in the metabolism of catecholamine neurotransmitters and is implicated in the modulation of cognitive and emotional responses. The best characterized single nucleotide polymorphism (SNP) of the COMT gene consists of a valine (Val)-to-methionine (Met) substitution at codon 108/158. The Met-containing variant confers a marked reduction in COMT catalytic activity. We recently showed that the activity of recombinant COMT is positively regulated by the enzyme Met sulphoxide reductase (MSR), which counters the oxidation of Met residues of proteins. The current study was designed to assess whether brain COMT activity may be correlated to MSR in an allele-dependent fashion. COMT and MSR activities were measured from post-mortem samples of prefrontal cortices, striata and cerebella of 32 subjects by using catechol and dabsyl-Met sulphoxide as substrates, respectively. Allelic discrimination of COMT Val(108/185) Met SNP was performed using the Taqman 5'nuclease assay. Our studies revealed that, in homozygous carriers of Met, but not Val alleles, the activity of COMT and MSR was significantly correlated throughout all tested brain regions. These results suggest that the reduced enzymatic activity of Met-containing COMT may be secondary to Met sulphoxidation and point to MSR as a key molecular determinant for the modulation of COMT activity. © 2015 British Neuropathological Society.

Enzymatic Hydrolysis of Pretreated Fibre Pressed Oil Palm Frond by using Sacchariseb C6

Science.gov (United States)

Hashim, F. S.; Yussof, H. W.; Zahari, M. A. K. M.; Rahman, R. A.; Illias, R. M.

2017-06-01

Enzymatic hydrolysis becomes a prominent technology for conversion of cellulosic biomass to its glucose monomers that requires an action of cellulolytic enzymes in a sequential and synergistic manner. In this study, the effect of agitation speed, glucan loading, enzyme loading, temperature and reaction time on the production of glucose from fibre pressed oil palm frond (FPOPF) during enzymatic hydrolysis was screened by a half factorial design 25-1 using Response Surface Methodology (RSM). The FPOPF sample was first delignified by alkaline pretreatment at 4.42 (w/v) sodium hydroxide for an hour prior to enzymatic hydrolysis using commercial cellulase enzyme, Sacchariseb C6. The effect of enzymatic hydrolysis on the structural of FPOPF has been evaluated by Scanning Electron Microscopy (SEM) analysis. Characterization of raw FPOPF comprised of 4.5 extractives, 40.7 glucan, 26.1 xylan, 26.2 lignin and 1.8 ash, whereas for pretreated FPOPF gave 0.3 extractives, 61.4 glucan, 20.4 xylan, 13.3 lignin and 1.3 ash. From this study, it was found that the best enzymatic hydrolysis condition yielded 33.01 ± 0.73 g/L of glucose when performed at 200 rpm of agitation speed, 60 FPU/mL of enzyme loading, 4 (w/w) of glucan loading, temperature at 55 □ and 72 hours of reaction time. The model obtained was significant with p-value enzymatic hydrolysis from pretreated FPOPF produce high amount of glucose that enhances it potential for industrial application. This glucose can be further used to produce high-value products.

Malondialdehyde level and some enzymatic activities in subclinical ...

African Journals Online (AJOL)

The purpose of this study was to evaluate the changes occurring in milk malondialdehyde (MDA) level and some enzymatic activities as a result of subclinical mastitis (SCM) in dairy cows. A total of 124 milk samples were collected from 124 lactating cows from the same herd in the period between the 2nd week after calving ...

Interleukin-6 enhances human Ig production, but not as a terminal differentiation factor for B lymphocytes

NARCIS (Netherlands)

van Kooten, C.; van Oers, M. H.; Aarden, L. A.

1990-01-01

Most B-cell differentiation systems are complicated by the fact that they are both T-cell- and monocyte-dependent. Immobilized anti-CD3 antibodies induce monocyte-independent T-cell activation, allowing investigation of the role of interleukin-6 (IL6) in the process of B-cell differentiation. We

Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

Science.gov (United States)

Heck, Julie A; Meng, Xiao; Frick, David N

2009-04-01

Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

Peroxiredoxin 6 expression is inversely correlated with nuclear factor-κB activation during Clonorchis sinensis infestation.

Science.gov (United States)

Pak, Jhang Ho; Son, Woo Chan; Seo, Sang-Beom; Hong, Sung-Jong; Sohn, Woon-Mok; Na, Byoung-Kuk; Kim, Tong-Soo

2016-10-01

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory-secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein β (C/EBPβ). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis. Copyright © 2016 Elsevier Inc. All rights reserved.

An Investigation of the Changes in Enzymatic and Non-Enzymatic Salivary Antioxidants Caused by Exhausting Aerobic Activity in Non-Athletic Men

Directory of Open Access Journals (Sweden)

Yazgaldi Nazari

2016-12-01

Full Text Available Background and Objectives: In the present study, the effect of acute aerobic exercise on enzymatic and non-enzymatic salivary antioxidants variations in non-athlete men, was investigated. Methods: In this experimental study, 25 male non-athlete collegiates (age, 21.2±1.6 years; weight, 68.62±10.1kg; body fat, 16.75±2.9%; and Vo2 max, 37.54±2.4ml/kg/min participated voluntarily in this study. Saliva samples were collected in three phases (before, immediately, and 1 hour after running on treadmill according to Astrand test. The activity of peroxidase and catalase, and concentration of uric acid were measured by laboratory methods. Then, to assess the obtained changes, repeated measures statistical test, and in case of significance, post-hoc Bonferroni test were used for pairwise comparing of the measuring phases at the significance level of p≤0.05 used.  Results: The activity of peroxidase significantly increased immediately and 1 hour after exercise compared to the baseline; Also, the concentration of uric acid significantly increased after aerobic exercise, but catalase enzyme activity significantly decreased after aerobic exercise (p<0.05. No significant change was observed in saliva flow rate after exercise. Conclusion: According to the findings of this study, aerobic exercise causes the production of free radicals, and salivary antioxidant system increases as the body biological response to neutralize and counteract the damaging effects of free radicals.

Is Oxidative Stress in Mice Brain Regions Diminished by 2-[(2,6-Dichlorobenzylideneamino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile?

Directory of Open Access Journals (Sweden)

A. C. Fortes

2013-01-01

Full Text Available 2-[(2,6-Dichlorobenzylideneamino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile, 5TIO1, is a new 2-aminothiophene derivative with promising pharmacological activities. The aim of this study was to evaluate its antioxidant activity in different areas of mice central nervous system. Male Swiss adult mice were intraperitoneally treated with Tween 80 dissolved in 0.9% saline (control group and 5TIO1 (0.1, 1, and 10 mg kg−1. Brain homogenates—hippocampus, striatum, frontal cortex, and cerebellum—were obtained after 24 h of observation. Superoxide dismutase and catalase activities, lipid peroxidation and nitrite content were measured using spectrophotometrical methods. To clarify the 5TIO1’s mechanism on oxidative stress, western blot analysis of superoxide dismutase and catalase was also performed. 5TIO1 decreased lipid peroxidation and nitrite content in all brain areas and increased the antioxidant enzymatic activities, specially, in cerebellum. The data of Western blot analysis did not demonstrate evidence of the upregulation of these enzymes after the administration of this compound. Our findings strongly support that 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies.

Differentiation of enzymatic activity of yeasts and yeast-like microorganisms isolated from various environments

Directory of Open Access Journals (Sweden)

Elżbieta Bogusławska-Wąs

2014-08-01

Full Text Available The aim of study was to determinate enzymatic activity of yeast-like organisms - Candida lipolytica, Rhodotorula rubra, Trichosporon beigelii, Zygosaccharomyces sp. - isolated from the Szczecin Lagoon and herring salads. We have shown that lipolytic activity was higher than protcolytic for every strain tested. The lowest activity level was found out for amylolytic hydrolases. The results also demonstrated that yeast-like organisms isolated from the Szczecin Lagoon revealed much higher average enzymatic activity compared to tbe same species isolated from herring salads, excepting C. lipolytica.

Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

Science.gov (United States)

Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

2014-07-01

Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

Science.gov (United States)

Kerr, T. P.

1985-01-01

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

Science.gov (United States)

Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

2010-08-01

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

Photoelectrochemical enzymatic biosensors.

Science.gov (United States)

Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2017-06-15

Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism

OpenAIRE

Valles, Soraya L; Benlloch, Mar?a; Rodriguez, Mar?a L; Mena, Salvador; Pellicer, Jos? A; Asensi, Miguel; Obrador, Elena; Estrela, Jos? M

2013-01-01

[EN] Background: Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms. Methods: Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic f...

Curcumin Regulates Low-Linear Energy Transfer γ-Radiation-Induced NFκB-Dependent Telomerase Activity in Human Neuroblastoma Cells

International Nuclear Information System (INIS)

Aravindan, Natarajan; Veeraraghavan, Jamunarani; Madhusoodhanan, Rakhesh; Herman, Terence S.; Natarajan, Mohan

2011-01-01

Purpose: We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. Methods and Materials: SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. Results: Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival. Conclusions: These results

PARTIAL CHARACTERIZATION OF ENZYMATIC ACTIVITIES PRODUCED BY A WILD STRAIN OF A. NIGER

Directory of Open Access Journals (Sweden)

María Martos

2012-12-01

Full Text Available Aspergillus niger, isolated from decay citrus peels in the province of Misiones, was able to produce pectinases by submerged fermentation. The enzymatic extract exhibited polygalacturonase, pectinesterase and lyase activities. Others enzymes capable of degrading cell wall polymers were also detected in the enzymatic extract such as cellulases and xylanases. Polygalacturonase was an endo-polygalacturonase. The enzyme exhibited a maximal activity at pH range between 4.5 to 5.0, was stable in the pH range from 2.5 to 5.5 and remained unchanged when was incubated at temperatures lower than 50 ºC. The fungi produced three PG isoenzymes. The enzymatic extract was able to clarify apple juice. The results observed make the pectinolytic enzymes produced by A. niger appropriate for future application in fruit juice processing industries.

Enzymatic Synthesis of Esculin Ester in Ionic Liquids Buffered with Organic Solvents

DEFF Research Database (Denmark)

Hu, Yifan; Guo, Zheng; Lue, Bena-Marie

2009-01-01

The enzymatic esterification of esculin catalyzed by Candida antarctica lipase B (Novozym 435) was carried out in ionic liquid (IL)-organic solvent mixed systems in comparison with individual systems. The reaction behaviors in IL-organic solvents were systemically evaluated using acetone as a model...... in IL-acetone mixtures made it possible to improve the solubility of esculin while the effects of ILs on lipase activity were minimized. Following the benignity of ILs to lipase activity, the anions of ILs were ranked in the order as [Tf2N](-) > [PF6](-) > [BF4](-) > [CF3SO3](-) > [C4F9SO3](-) > [TAF...

HPLC analysis of vitamin B6 in foods

Directory of Open Access Journals (Sweden)

V.-M. OLLILAINEN

2008-12-01

Full Text Available The objective of this work was to evaluate the methods for determination of vitamin B6 in foods. To achieve this, the literature review focused on sample treatment and liquid chromatographic analysis of vitamin B6 related compounds. In the experimental part, the chosen sample pretreatment and the high-performance liquid chromatographic (HPLC method were validated, and used to produce vitamin B6 data on various food items commonly consumed in Finland. The main emphasis of the sample treatment was on the extraction efficiency and the maintenance of the original concentration profile of the vitamers. Several acid extraction procedures were tested for this purpose. Perchloric acid was chosen as the extraction agent. Routine food analysis was then performed using dilute ice-cold perchloric acid extraction followed by an internally standardized ion-paired reversed-phase liquid chromatography. Food samples were hydrolyzed with takadiastase and alkaline phosphatase enzymes, phosphorylated and glycosylated vitamers were quantitated before and after the enzymatic digestion. This procedure enabled the extraction of vitamin B6 compounds in their intact forms, and the measurement of free, phosphorylated and glycosylated forms. The maintenance of the concentration profile of the vitamers was verified by using 14C -labeled pyridoxal 5'-phosphate in the examination of the extraction procedure. The extraction efficiency and laboratory performance were confirmed by interlaboratory studies. Up-to-date data on vitamin B6 content of about fifty common food items was produced. The data includes the results from meat and poultry, fish and fish product, dairy product, cereal and vegetable, and ready-to-eat food samples. Free and phosphorylated vitamin B6 compounds were measured in all food groups, and the glycosylated vitamer fraction was analyzed in all plant-derived foods. The results obtained in this work showed that vitamin B6 content of nearly all foods of plant

Enzymatic Activity of Candida spp. from Oral Cavity and Urine in Children with Nephrotic Syndrome.

Science.gov (United States)

Olczak-Kowalczyk, Dorota; Roszkowska-Blaim, Maria; Dąbkowska, Maria; Swoboda-Kopeć, Ewa; Gozdowski, Dariusz; Mizerska-Wasiak, Małgorzata; Demkow, Urszula; Pańczyk-Tomaszewska, Małgorzata

2017-01-01

Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.

Induction of mast cell accumulation by chymase via an enzymatic activity- and intercellular adhesion molecule-1-dependent mechanism.

Science.gov (United States)

Zhang, Huiyun; Wang, Junling; Wang, Ling; Zhan, Mengmeng; Li, Shigang; Fang, Zeman; Xu, Ciyan; Zheng, Yanshan; He, Shaoheng

2018-02-01

Chymase is a unique, abundant secretory product of mast cells and a potent chemoattractant for eosinophils, monocytes and neutrophils, but little is known of its influence on mast cell accumulation. A mouse peritoneal inflammation model, cell migration assay and flowcytometry analysis, were used to investigate the role of chymase in recruiting mast cells. Chymase increased, by up to 5.4-fold, mast cell numbers in mouse peritoneum. Inhibitors of chymase, heat-inactivation of the enzyme, sodium cromoglycate and terfenadine, and pretreatment of mice with anti-intercellular adhesion molecule 1, anti-L-selectin, anti-CD11a and anti-CD18 antibodies dramatically diminished the chymase-induced increase in mast cell accumulation. These findings indicate that this effect of chymase is dependent on its enzymatic activity and activation of adhesion molecules. In addition, chymase provoked a significant increase in 5-HT and eotaxin release (up to 1.8- and 2.2-fold, respectively) in mouse peritoneum. Since 5-HT, eotaxin and RANTES can induce marked mast cell accumulation, these indirect mechanisms may also contribute to chymase-induced mast cell accumulation. Moreover, chymase increased the trans-endothelium migration of mast cells in vitro indicating it also acts as a chemoattractant. The finding that mast cells accumulate in response to chymase implies further that chymase is a major pro-inflammatory mediator of mast cells. This effect of chymase, a major product of mast cell granules, suggests a novel self-amplification mechanism for mast cell accumulation in allergic inflammation. Mast cell stabilizers and inhibitors of chymase may have potential as a treatment of allergic disorders. © 2017 The British Pharmacological Society.

Radiation pretreatments of cellulose materials for the enhancement of enzymatic hydrolysis

International Nuclear Information System (INIS)

Ardica, S.; Calderaro, E.; Cappadona, C.

1985-01-01

The effect of γ-ray pre-irradiation of cellulose materials such as wood chips, paper, grain straw, hay and kapok on glucose production on enzymatic hydrolysis by cellulase has been investigated. These materials have been irradiated in air, water and acetate buffer solution over the dose range 10 3 to 4 x 10 6 Gy. In the relatively low dose range, up to about 5 x 10 5 Gy, the glucose yields after enzymatic hydrolysis are practically insensitive to radiation. At higher dose levels, up to 1.7 to 2 x 10 6 Gy, the pre-irradiation becomes very effective on enzymatic cellulose conversion. It has been found that the radiation-induced degradation of cellulose into low molecular weight polysaccharides is dependent on the nature and chemical composition of the cellulose materials and on the radiation environmental conditions. Further increases of dose causes radiation-induced structural modifications in polysaccharides previously produced, which can lead to a decrease in glucose production by enzymatic hydrolysis. (author)

HPLC analysis of water-soluble vitamins (B1, B2, B3, B5, B6) in in ...

African Journals Online (AJOL)

... B6 (pyridoxine HCl) were higher in in vitro germinated seedlings. Thus, vitamin production was age and culture conditions dependent, which is discussed in detail. The study revealed that the germinated chickpea grains can be used for human consumption with value addition of vitamin B-group in the diet of vegetarians.

E. coli-Derived L-Asparaginase Retains Enzymatic and Cytotoxic Activity In Vitro for Canine and Feline Lymphoma after Cold Storage

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Jackie M. Wypij

2013-01-01

Full Text Available Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets. Hypothesis. E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage. Methods. E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days and freezing (14 days–six months, one to three freeze/thaw cycles. Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B and canine (17–71, OSW lymphoma cells. Results. Cold-stored E. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-stored E. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity. Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstituted E. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility of E. coli-derived asparaginase as a therapeutic option for pets with lymphoma.

Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone

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Eun Chae Gong

2018-03-01

Full Text Available Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae, exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb–drug interaction (HDI through cytochrome P450s (CYPs-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki values of 14.3, 16.8, 41.7, and 6.84 μM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone–drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

Effects of UV-B irradiation on isoforms of antioxidant enzymes and their activities in red alga Grateloupia filicina (Rhodophyta)

Science.gov (United States)

Zhao, Jiqiang; Li, Lixia

2014-11-01

Macroalgae in a littoral zone are inevitably exposed to UV-B irradiance. We analyzed the effects of UV-B on isoenzyme patterns and activities of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) of red algae Grateloupia filicina (Lamour.) C. Agardh. The activities of SOD, CAT, and APX changed in response to UV-B in a time- and dose-dependent manner. POX activity increased significantly under all three UV-B treatments. The enzymatic assay showed three distinct bands of SODI (Mn-SOD), SODII (Fe-SOD), and SODIII (CuZn-SOD) under a low (Luv) and medium (Muv) dose of UV-B irradiation, while SODI and SODIII activities decreased significantly when exposed to a high dose of UV-B irradiation (Huv). The activity of POX isoenzymes increased significantly after exposure to UV-B, which is consistent with the total activity. In addition, a clear decrease in activity of CATIV was detected in response to all the three doses of UV treatments. Some bands of APX isoenzyme were also clearly influenced by UV-B irradiation. Correspondingly, the daily growth rate declined under all the three exposure doses, and was especially significant under Muv and Huv treatments. These data suggest that, although the protection mechanisms of antioxidant defense system are partly inducible by UV-B to prevent the damage, G. filicina has incomplete tolerance to higher UV-B irradiation stress.

Key Amino Acids in the Bacterial (6-4) Photolyase PhrB from Agrobacterium fabrum.

Science.gov (United States)

Graf, Dominik; Wesslowski, Janine; Ma, Hongju; Scheerer, Patrick; Krauß, Norbert; Oberpichler, Inga; Zhang, Fan; Lamparter, Tilman

2015-01-01

Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4) photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4) photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4) photolyases.

Key Amino Acids in the Bacterial (6-4 Photolyase PhrB from Agrobacterium fabrum.

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Dominik Graf

Full Text Available Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4 photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4 photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4 photolyases.

Akt-dependent NF-κB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis

International Nuclear Information System (INIS)

Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S.; Wang Jianying; Raufman, Jean-Pierre

2009-01-01

Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-κB. Deoxycholyltaurine (DCT) treatment attenuated TNF-α-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-κB nuclear translocation and transcriptional activity (detected by NF-κB binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-κB nuclear translocation and attenuation of TNF-α-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and IκBα kinase attenuated NF-κB transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable IκBα 'super-repressor' blocked the actions of DCT on both NF-κB activation and TNF-α-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-κB transcriptional activity and TNF-α-induced apoptosis. Chemical inhibitors of Akt and NF-κB activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-κB activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation

Impact of orientation of carbohydrate binding modules family 22 and 6 on the catalytic activity of Thermotoga maritima xylanase XynB.

Science.gov (United States)

Tajwar, Razia; Shahid, Saher; Zafar, Rehan; Akhtar, Muhammad Waheed

2017-11-01

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before. Copyright © 2017 Elsevier Inc. All rights reserved.

Coexistence of metallic and insulating channels in compressed YbB6

Science.gov (United States)

Ying, Jianjun; Tang, Lingyun; Chen, Fei; Chen, Xianhui; Struzhkin, Viktor V.

2018-03-01

It remains controversial whether compressed YbB6 material is a topological insulator or a Kondo topological insulator. We performed high-pressure transport, x-ray diffraction (XRD), x-ray absorption spectroscopy, and Raman-scattering measurements on YbB6 samples in search for its topological Kondo phase. Both high-pressure powder XRD and Raman measurements show no trace of structural phase transitions in YbB6 up to 50 GPa. The nonmagnetic Yb2 + gradually change to magnetic Yb3 + above 18 GPa concomitantly with the increase in resistivity. However, the transition to the insulating state occurs only around 30 GPa, accompanied by the increase in the shear stress, and anomalies in the pressure dependence of the Raman T2 g mode and in the B atomic position. The resistivity at high pressures can be described by a model taking into account coexisting insulating and metallic channels with the activation energy for the insulating channel about 30 meV. We argue that YbB6 may become a topological Kondo insulator at high pressures above 35 GPa.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

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Neal J Dawson

Full Text Available One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05. A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively. Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

Science.gov (United States)

Dawson, Neal J; Biggar, Kyle K; Storey, Kenneth B

2013-01-01

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Enzymatic biomarkers can portray nanoCuO-induced oxidative and neuronal stress in freshwater shredders.

Science.gov (United States)

Pradhan, Arunava; Silva, Carla O; Silva, Carlos; Pascoal, Cláudia; Cássio, Fernanda

2016-11-01

Commercial applications of nanometal oxides have increased concern about their release into natural waters and consequent risks to aquatic biota and the processes they drive. In forest streams, the invertebrate shredder Allogamus ligonifer plays a key role in detritus food webs by transferring carbon and energy from plant litter to higher trophic levels. We assessed the response profiles of oxidative and neuronal stress enzymatic biomarkers in A. ligonifer after 96h exposure to nanoCuO at concentration ranges stress, Cu 2+ released from nanoCuO was quantified and the enzymatic responses to Cu 2+ exposure at similar effective concentrations were compared. The highest activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed at concentrations stress at low concentrations (released ionic copper on enzyme activities were concentration-dependent, and led to oxidative stress and even to animal death. The activity of acetylcholinesterase (AChE) was strongly inhibited even at concentrations stress in A. ligonifer. Copyright © 2016 Elsevier B.V. All rights reserved.

Methylation-Dependent Activation of CDX1 through NF-κB

Science.gov (United States)

Rau, Tilman T.; Rogler, Anja; Frischauf, Myrjam; Jung, Andreas; Konturek, Peter C.; Dimmler, Arno; Faller, Gerhard; Sehnert, Bettina; El-Rifai, Wael; Hartmann, Arndt; Voll, Reinhard E.; Schneider-Stock, Regine

2013-01-01

The caudal homeobox factor 1 (CDX1) is an essential transcription factor for intestinal differentiation. Its aberrant expression in intestinal metaplasia of the upper gastrointestinal tract is a hallmark within the gastritis-metaplasia-carcinoma sequence. CDX1 expression is influenced by certain pathways, such as Wnt, Ras, or NF-κB signaling; however, these pathways alone cannot explain the transient expression of CDX1 in intestinal metaplasia or the molecular inactivation mechanism of its loss in cases of advanced gastric cancer. In this study, we investigated the epigenetic inactivation of CDX1 by promoter methylation, as well as the functional link of CDX1 promoter methylation to the inflammatory NF-κB signaling pathway. We identified methylation-dependent NF-κB binding to the CDX1 promoter and quantified it using competitive electrophoretic mobility shift assays and chromatin immunoprecipitation. A methylated CDX1 promoter was associated with closed chromatin structure, reduced NF-κB binding, and transcriptional silencing. Along the gastritis-metaplasia-carcinoma sequence, we observed a biphasic pattern of tumor necrosis factor-α (TNF-α) protein expression and an inverse biphasic pattern of CDX1 promoter methylation; both are highly consistent with CDX1 protein expression. The stages of hyper-, hypo-, and hyper-methylation patterns of the CDX1 promoter were inversely correlated with the NF-κB signaling activity along this sequence. In conclusion, these functionally interacting events drive CDX1 expression and contribute to intestinal metaplasia, epithelial dedifferentiation, and carcinogenesis in the human stomach. PMID:22749770

New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel†

Science.gov (United States)

Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

2012-01-01

The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation–anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym® 435 in choline acetate/glycerol (1 : 1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel. PMID:21283901

Rare ginsenoside Ia synthesized from F1 by cloning and overexpression of the UDP-glycosyltransferase gene from Bacillus subtilis: synthesis, characterization, and in vitro melanogenesis inhibition activity in BL6B16 cells.

Science.gov (United States)

Wang, Dan-Dan; Jin, Yan; Wang, Chao; Kim, Yeon-Ju; Perez, Zuly Elizabeth Jimenez; Baek, Nam In; Mathiyalagan, Ramya; Markus, Josua; Yang, Deok-Chun

2018-01-01

Ginsenoside F1 has been described to possess skin-whitening effects on humans. We aimed to synthesize a new ginsenoside derivative from F1 and investigate its cytotoxicity and melanogenesis inhibitory activity in B16BL6 cells using recombinant glycosyltransferase enzyme. Glycosylation has the advantage of synthesizing rare chemical compounds from common compounds with great ease. UDP-glycosyltransferase (BSGT1) gene from Bacillus subtilis was selected for cloning. The recombinant glycosyltransferase enzyme was purified, characterized, and utilized to enzymatically transform F1 into its derivative. The new product was characterized by NMR techniques and evaluated by MTT, melanin count, and tyrosinase inhibition assay. The new derivative was identified as (20 S )-3 β ,6 α ,12 β ,20-tetrahydroxydammar-24-ene-20- O - β -D-glucopyranosyl-3- O - β -D-glucopyranoside (ginsenoside Ia), which possesses an additional glucose linked into the C-3 position of substrate F1. Ia had been previously reported; however, no in vitro biological activity was further examined. This study focused on the mass production of arduous ginsenoside Ia from accessible F1 and its inhibitory effect of melanogenesis in B16BL6 cells. Ia showed greater inhibition of melanin and tyrosinase at 100 μmol/L than F1 and arbutin. These results suggested that Ia decreased cellular melanin synthesis in B16BL6 cells through downregulation of tyrosinase activity. To our knowledge, this is the first study to report on the mass production of rare ginsenoside Ia from F1 using recombinant UDP-glycosyltransferase isolated from B. subtillis and its superior melanogenesis inhibitory activity in B16BL6 cells as compared to its precursor. In brief, ginsenoside Ia can be applied for further study in cosmetics.

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

International Nuclear Information System (INIS)

Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

2011-01-01

Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

Energy Technology Data Exchange (ETDEWEB)

Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

2011-08-05

Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

Science.gov (United States)

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

Directory of Open Access Journals (Sweden)

Rami Gherib

2013-12-01

Full Text Available Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis.

Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose; Actividad enzimatica del complejo celulolitico producido por Trichoderma reesei. Hidrolisis enzimatica de la celulosa

Energy Technology Data Exchange (ETDEWEB)

Alfonsel, M; Negro, M J; Saez, R; Martin, C

1986-07-01

The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs.

Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

Science.gov (United States)

Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

2013-12-01

This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like. © 2013. Published by Elsevier B.V. All rights reserved.

Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target?

Directory of Open Access Journals (Sweden)

Thales Kronenberger

2014-01-01

Full Text Available Malaria is a deadly infectious disease which affects millions of people each year in tropical regions. There is no effective vaccine available and the treatment is based on drugs which are currently facing an emergence of drug resistance and in this sense the search for new drug targets is indispensable. It is well established that vitamin biosynthetic pathways, such as the vitamin B6 de novo synthesis present in Plasmodium, are excellent drug targets. The active form of vitamin B6, pyridoxal 5-phosphate, is, besides its antioxidative properties, a cofactor for a variety of essential enzymes present in the malaria parasite which includes the ornithine decarboxylase (ODC, synthesis of polyamines, the aspartate aminotransferase (AspAT, involved in the protein biosynthesis, and the serine hydroxymethyltransferase (SHMT, a key enzyme within the folate metabolism.

Immobilization of Lipase using Alginate Hydrogel Beads and Enzymatic Evaluation in Hydrolysis of p-Nitrophenol Butyrate

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Zhang, Shuang; Shang, Wenting; Yang, Xiaoxi; Zhang, Shujuan; Zhang, Xiaogang; Chen, Jiawei [Renmin Univ. of China, Beijing (China)

2013-09-15

The immobilization of enzyme is one of the key issues both in the field of enzymatic research and industrialization. In this work, we reported a facile method to immobilize Candida Antarctica lipase B (CALB) in alginate carrier. In the presence of calcium cation, the enzyme-alginate suspension could be cross-linked to form beads with porous structure at room temperature, and the enzyme CALB was dispersed in the beads. Activity of the enzyme-alginate composite was verified by enzymatic hydrolysis reaction of p-nitrophenol butyrate in aqueous phase. The effects of reaction parameters such as temperature, pH, embedding and lyophilized time on the reactive behavior were discussed. Reuse cycle experiments for the hydrolysis of p-nitrophenol butyrate demonstrated that activity of the enzyme-alginate composite was maintained without marked deactivation up to 6 repeated cycles.

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

Science.gov (United States)

Chourashi, Rhishita; Das, Suman; Dhar, Debarpan; Okamoto, Keinosuke; Mukhopadhyay, Asish K; Chatterjee, Nabendu Sekhar

2018-05-01

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Vitamin B6

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... Consumer Datos en español Health Professional Other Resources Vitamin B6 Fact Sheet for Consumers Have a question? ... out more about vitamin B6? Disclaimer What is vitamin B6 and what does it do? Vitamin B6 ...

PARP1 Val762Ala polymorphism reduces enzymatic activity

International Nuclear Information System (INIS)

Wang Xiaogan; Wang Zhaoqi; Tong Weimin; Shen Yan

2007-01-01

Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K m of PARP1-Ala762 was increased to a 1.2-fold of the K m of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K m . This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy*

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Matzner, Ulrich; Breiden, Bernadette; Schwarzmann, Günter; Yaghootfam, Afshin; Fluharty, Arvan L.; Hasilik, Andrej; Sandhoff, Konrad; Gieselmann, Volkmar

2009-01-01

Arylsulfatase A (ASA) catalyzes the intralysosomal desulfation of 3-O-sulfogalactosylceramide (sulfatide) to galactosylceramide. The reaction requires saposin B (Sap B), a non-enzymatic proteinaceous cofactor which presents sulfatide to the catalytic site of ASA. The lack of either ASA or Sap B results in a block of sulfatide degradation, progressive intralysosomal accumulation of sulfatide, and the fatal lysosomal storage disease metachromatic leukodystrophy. We studied the coupled Sap B-ASA reaction in vitro using detergent-free micellar and liposomal assay systems and in vivo using cell culture models of metachromatic leukodystrophy. Under in vitro conditions, the reaction had a narrow pH optimum around pH 4.3 and was inhibited by mono- and divalent cations, phosphate and sulfite. Bis(monoacylglycero) phosphate and phosphatidic acid were activators of the reaction, underscoring a significant role of acidic phosphoglycerolipids in sphingolipid degradation. Desulfation was negligible when Sap B was substituted by Sap A, C, or D. Up to a molar ratio between Sap B and sulfatide of 1:5, an elevation of Sap B concentrations caused a sharp increase of sulfatide hydrolysis, indicating the requirement of unexpected high Sap B levels for maximum turnover. Feeding of ASA-deficient, sulfatide-storing primary mouse kidney cells with ASA caused partial clearance of sulfatide. Co-feeding of Sap B or its precursor prosaposin resulted in the lysosomal uptake of the cofactor but did not promote ASA-catalyzed sulfatide hydrolysis. This suggests that Sap B is not a limiting factor of the coupled Sap B-ASA reaction in mouse kidney cells even if sulfatide has accumulated to unphysiologically high levels. PMID:19224915

TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

Science.gov (United States)

Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

2016-05-01

Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9. © Society for Leukocyte Biology.

A Method for the Determination of Bi-substrate Kinetic Coefficients: the Example of the b-D-glucose- NAD-GDH Enzymatic Reaction

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Jean BERTHIER

2015-08-01

Full Text Available Colorimetric detection of glucose in sample liquids such as human plasma is made by using enzymatic reactions. Either glucose oxidase (GOX or glucose dehydrogenase (GDH can be used to convert glucose. In the multi reactional scheme, the first enzymatic reaction is determinant. We focused here on the study of the enzyme GDH together with the enzymatic cofactor NAD (nicotinamide adenine dinucleotide. This reaction falls in the category of ternary enzymatic reactions. Such reactions depend on four parameters. A method to determine these four parameters is presented in this work, based on a comparison between a series of experiments and the theory. The best values of the parameters are indicated.

The development of orally administrable gemcitabine prodrugs with D-enantiomer amino acids: enhanced membrane permeability and enzymatic stability.

Science.gov (United States)

Tsume, Yasuhiro; Incecayir, Tuba; Song, Xueqin; Hilfinger, John M; Amidon, Gordon L

2014-04-01

Gemcitabine prodrugs with D- and L-configuration amino acids were synthesized and their chemical stability in buffers, resistance to glycosidic bond metabolism, enzymatic activation, permeability in Caco-2 cells and mouse intestinal membrane, anti-proliferation activity in cancer cell were determined and compared to that of parent drug, gemcitabine. Prodrugs containing D-configuration amino acids were enzymatically more stable than ones with L-configuration amino acids. The activation of all gemcitabine prodrugs was 1.3-17.6-fold faster in cancer cell homogenate than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of amino acid monoester prodrugs containing D-configuration amino acids in cell homogenates was 2.2-10.9-fold slower than one of amino acid monoester prodrugs with L-configuration amino acids. All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent gemcitabine. Gemcitabine prodrugs showed superior the effective permeability in mouse jejunum to gemcitabine. More importantly, the high plasma concentration of d-amino acid gemcitabine prodrugs was observed more than one of L-amino acid gemcitabine prodrugs. In general, the 5'-mono-amino acid monoester gemcitabine prodrugs exhibited higher permeability and uptake than their parent drug, gemcitabine. Cell proliferation assays in AsPC-1 pancreatic ductal cell line indicated that gemcitabine prodrugs were more potent than their parent drug, gemcitabine. The transport and enzymatic profiles of 5'-D-valyl-gemcitabine and 5'-D-phenylalanyl-gemcitabine suggest their potential for increased oral uptake and delayed enzymatic bioconversion as well as enhanced uptake and cytotoxic activity in cancer cells, would facilitate the development of oral dosage form for anti-cancer agents and, hence, improve the quality of life for the cancer patients. Copyright © 2014. Published by Elsevier B.V.

XEDAR activates the non-canonical NF-κB pathway

International Nuclear Information System (INIS)

Verhelst, Kelly; Gardam, Sandra; Borghi, Alice; Kreike, Marja; Carpentier, Isabelle; Beyaert, Rudi

2015-01-01

Members of the tumor necrosis factor receptor (TNFR) superfamily are involved in a number of physiological and pathological responses by activating a wide variety of intracellular signaling pathways. The X-linked ectodermal dysplasia receptor (XEDAR; also known as EDA2R or TNFRSF27) is a member of the TNFR superfamily that is highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2), a member of the TNF family that is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Although XEDAR was first described in the year 2000, its function and molecular mechanism of action is still largely unclear. XEDAR has been reported to activate canonical nuclear factor κB (NF-κB) signaling and mitogen-activated protein (MAP) kinases. Here we report that XEDAR is also able to trigger the non-canonical NF-κB pathway, characterized by the processing of p100 (NF-κB2) into p52, followed by nuclear translocation of p52 and RelB. We provide evidence that XEDAR-induced p100 processing relies on the binding of XEDAR to TRAF3 and TRAF6, and requires the kinase activity of NIK and IKKα. We also show that XEDAR stimulation results in NIK accumulation and that p100 processing is negatively regulated by TRAF3, cIAP1 and A20. - Highlights: • XEDAR activates the non-canonical NF-κB pathway. • XEDAR-induced processing of p100 depends on XEDAR interaction with TRAF3 and TRAF6. • XEDAR-induced processing of p100 depends on NIK and IKKα activity. • Overexpression of XEDAR leads to NIK accumulation. • XEDAR-induced processing of p100 is negatively regulated by TRAF3 cIAP1 and A20

XEDAR activates the non-canonical NF-κB pathway

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Verhelst, Kelly, E-mail: Kelly.Verhelst@irc.VIB-UGent.be [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium); Gardam, Sandra, E-mail: s.gardam@garvan.org.au [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium); Borghi, Alice, E-mail: Alice.Borghi@irc.VIB-UGent.be [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium); Kreike, Marja, E-mail: Marja.Kreike@irc.VIB-UGent.be [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium); Carpentier, Isabelle, E-mail: Isabelle.Carpentier@irc.VIB-UGent.be [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium); Beyaert, Rudi, E-mail: Rudi.Beyaert@irc.VIB-UGent.be [Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent (Belgium); Department of Biomedical Molecular Biology, Ghent University, Ghent (Belgium)

2015-09-18

Members of the tumor necrosis factor receptor (TNFR) superfamily are involved in a number of physiological and pathological responses by activating a wide variety of intracellular signaling pathways. The X-linked ectodermal dysplasia receptor (XEDAR; also known as EDA2R or TNFRSF27) is a member of the TNFR superfamily that is highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2), a member of the TNF family that is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Although XEDAR was first described in the year 2000, its function and molecular mechanism of action is still largely unclear. XEDAR has been reported to activate canonical nuclear factor κB (NF-κB) signaling and mitogen-activated protein (MAP) kinases. Here we report that XEDAR is also able to trigger the non-canonical NF-κB pathway, characterized by the processing of p100 (NF-κB2) into p52, followed by nuclear translocation of p52 and RelB. We provide evidence that XEDAR-induced p100 processing relies on the binding of XEDAR to TRAF3 and TRAF6, and requires the kinase activity of NIK and IKKα. We also show that XEDAR stimulation results in NIK accumulation and that p100 processing is negatively regulated by TRAF3, cIAP1 and A20. - Highlights: • XEDAR activates the non-canonical NF-κB pathway. • XEDAR-induced processing of p100 depends on XEDAR interaction with TRAF3 and TRAF6. • XEDAR-induced processing of p100 depends on NIK and IKKα activity. • Overexpression of XEDAR leads to NIK accumulation. • XEDAR-induced processing of p100 is negatively regulated by TRAF3 cIAP1 and A20.

Poxviral protein A52 stimulates p38 mitogen-activated protein kinase (MAPK) activation by causing tumor necrosis factor receptor-associated factor 6 (TRAF6) self-association leading to transforming growth factor β-activated kinase 1 (TAK1) recruitment.

Science.gov (United States)

Stack, Julianne; Hurst, Tara P; Flannery, Sinead M; Brennan, Kiva; Rupp, Sebastian; Oda, Shun-ichiro; Khan, Amir R; Bowie, Andrew G

2013-11-22

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Radioimmunoassay of inactive creatine kinase B protein in human plasma

Energy Technology Data Exchange (ETDEWEB)

Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

1981-08-27

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

Radioimmunoassay of inactive creatine kinase B protein in human plasma

International Nuclear Information System (INIS)

Burnam, M.H.; Shell, W.E.

1981-01-01

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

Erythrocyte vitamin B, and B6 dependent enzymes in adult and newborn Thoroughbred horses

Directory of Open Access Journals (Sweden)

Maria de Fátima Cepa Matos

1996-09-01

Full Text Available Com o propósito de determinar o estado nutricional de cavalos Puro-Sangue Inglês (PSI em relação à riboflavina (vitamina B2 e à piridoxina (vitamina B6, 10 animais adultos e 30 recém-nascidos foram investigados. Foi observado um bom estado nutricional quanto à riboflavina, notando-se moderada deficiência de piridoxina nos animais adultos, mas não nos recém-nascidos. Estes fatos sugerem que os animais adultos devam receber suplementação com piridoxina.

Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.

Science.gov (United States)

Ndiaye, Fatou; Vuong, Tri; Duarte, Jairo; Aluko, Rotimi E; Matar, Chantal

2012-02-01

Enzymatic protein hydrolysates of yellow pea seed have been shown to possess high anti-oxidant and anti-bacterial activities. The aim of this work was to confirm the anti-oxidant, anti-inflammatory and immunomodulating activities of an enzymatic protein hydrolysate of yellow field pea seeds. The anti-oxidant and anti-inflammatory properties of peptides from yellow field pea proteins (Pisum sativum L.) were investigated in LPS/IFN-γ-activated RAW 264.7 NO⁻ macrophages. The immunomodulating potential of pea protein hydrolysate (PPH) was then studied in a murine model. Pea protein hydrolysate, after a 12 h pre-treatment, showed significant inhibition of NO production by activated macrophages up to 20%. Moreover, PPH significantly inhibited their secretion of pro-inflammatory cytokines, TNF-α- and IL-6, up to 35 and 80%, respectively. Oral administration of PPH in mice enhanced the phagocytic activity of their peritoneal macrophages and stimulated the gut mucosa immune response. The number of IgA+ cells was elevated in the small intestine lamina propria, accompanied by an increase in the number of IL-4+, IL-10+ and IFN-γ+ cells. This was correlated to up-regulation of IL-6 secretion by small intestine epithelial cells (IEC), probably responsible for B-cell terminal differentiation to IgA-secreting cells. Moreover, PPH might have increased IL-6 production in IECs via the stimulation of toll-like receptors (TLRs) family, especially TLR2 and TLR4 since either anti-TLR2 or anti-TLR4 was able to completely abolish PPH-induced IL-6 secretion. Enzymatic protein degradation confers anti-oxidant, anti-inflammatory and immunomodulating potentials to pea proteins, and the resulted peptides could be used as an alternative therapy for the prevention of inflammatory-related diseases.

Effects of clopidogrel and clarithromycin on the disposition of sibutramine and its active metabolites M1 and M2 in relation to CYP2B6*6 polymorphism.

Science.gov (United States)

Pan, Wei; Bae, Soo-Kyung; Shim, Eon-Jeong; Park, Sung-Eun; Lee, Sang-Seop; Park, Soo-Jin; Yeo, Chang-Woo; Zhou, Hong-Hao; Shon, Ji-Hong; Shin, Jae-Gook

2013-02-01

Plasma concentrations of sibutramine and its two active metabolites after single oral dose of sibutramine were determined in Korean healthy male subjects with different CYP2B6 genotypes (CYP2B6*1/*1, *1/*6 and *6/*6), either alone or after four-day pretreatment with clopidogrel or clarithromycin. The pretreatment with clopidogrel and clarithromycin raised the mean area under the concentration-time curve (AUC) of sibutramine by 163% and 255%, respectively. Co-administration of clarithromycin, combined with CYP2B6*6/*6 genotype, led to highest concentration of sibutramine. The molar sum AUC (M1 + M2) was raised by 35% in the clopidogrel phase but not significantly affected by clarithromycin or CYP2B6 genotype. The CYP2B6*6/*6 subjects in the clopidogrel phase showed the highest molar AUC (M1 + M2) among three genotype groups throughout the three phases. The exposure of sibutramine and its metabolites seemed to be associated with the CYP2B6 genotype. The treatment of clopidogrel significantly altered the disposition of active metabolites as well as sibutramine, but clarithromycin only affects the disposition of sibutramine. These results suggest that the perturbation of CYP2B6 activity may contribute to the inter-individual variation of sibutramine drug responses although the clinical relevance is remained to be established.

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

International Nuclear Information System (INIS)

Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

2005-01-01

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

Heterologous expression of Helicoverpa armigera cytochrome P450 CYP6B7 in Pichia pastoris and interactions of CYP6B7 with insecticides.

Science.gov (United States)

Zhao, Chunqing; Song, Genmiao; Duan, Hongxia; Tang, Tao; Wang, Chen; Qiu, Lihong

2017-09-01

Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot. Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively. The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

International Nuclear Information System (INIS)

Han, Seong-Su; Tompkins, Van S.; Son, Dong-Ju; Kamberos, Natalie L.; Stunz, Laura L.; Halwani, Ahmad; Bishop, Gail A.; Janz, Siegfried

2013-01-01

Highlights: •Mouse model of human Burkitt lymphoma revealed cancer inhibition by PL. •Treatment with PL led to apoptosis of malignant but not normal B cells. •PL inhibited LMP1–NF-κB–Myc-dependent target genes including p21-encoding Cdkn1a. •PL holds promise for new interventions approaches to hematologic malignancies. -- Abstract: Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc Eμ . PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21 Cip1 -encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers

Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

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Han, Seong-Su; Tompkins, Van S. [Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Son, Dong-Ju [Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Kamberos, Natalie L. [Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Stunz, Laura L. [Deparment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Iowa City VAMC, Iowa City, IA (United States); Halwani, Ahmad [Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Bishop, Gail A. [Deparment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Iowa City VAMC, Iowa City, IA (United States); Janz, Siegfried, E-mail: siegfried-janz@uiowa.edu [Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA (United States)

2013-07-12

Highlights: •Mouse model of human Burkitt lymphoma revealed cancer inhibition by PL. •Treatment with PL led to apoptosis of malignant but not normal B cells. •PL inhibited LMP1–NF-κB–Myc-dependent target genes including p21-encoding Cdkn1a. •PL holds promise for new interventions approaches to hematologic malignancies. -- Abstract: Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc{sup Eμ}. PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21{sup Cip1}-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers.

The dependence of the discharge of nitrous oxide by ordinary chernozem steppe of the Central-Chernozem Region of Russia from the content of humus, nitrogen and enzymatic activity

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Avksentev, Alexey; Negrobova, Elena; Kramareva, Tatiana; Moiseeva, Evgenya

2016-04-01

The dependence of the discharge of nitrous oxide by ordinary chernozem steppe of the Central-Chernozem Region of Russia from the content of humus, nitrogen and enzymatic activity Alexey Avksentev, Elena Negrobova, Tatiana Kramareva, Evgenya Moiseeva 394000 Voronezh, Universitetskaya square, 1 Voronezh State University Nitrous oxide is emitted by soil as a result of microbiological processes, ranks third in the list of aggressive greenhouse gas after carbon dioxide and methane. Nitrous oxide is formed during nitrification and denitrification of ammonia that enters the soil during microbial decomposition of complex organic compounds. Denitrification can be direct and indirect. In the microbiological process of recovery of nitrates involved of the organic substance. In aerobic conditions microorganisms denitrificator behave like normal saprotrophs and oxidize organic matter in the act of breathing oxygen. Thus, they operate at different times two enzyme systems: the electron transport chain with an oxygen acceptor in aerobic and restoration of nitrates under anaerobic conditions. Investigation of the emission of nitrous oxide by ordinary Chernozem steppe of the Central-Chernozem Region showed that it depends on the type of cenosis and the content of available forms of nitrogen. Natural ecosystems emit nitrous oxide more than the soil of arable land. The dependence of the emission of nitrous oxide from the humus content shows positive trend, but the aggregation of data, significant differences are not detected. Research shows that nitrous oxide emissions are seasonal. So the autumn season is characterized by nitrous oxide emissions than spring. Enzymatic processes are an important link in the biological cycle of elements and, consequently, participate in the process of decomposition of organic matter, nitrification and other processes. Analysis of the data on enzyme activity of ordinary Chernozem and the intensity of emission of N20 shows a clear relationship between

Polymorphisms of cytochrome P450 2B6 (CYP2B6) in cynomolgus and rhesus macaques.

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Uno, Yasuhiro; Uehara, Shotaro; Yamazaki, Hiroshi

2018-02-22

Cytochrome P450 2B6 (CYP2B6) is an important drug-metabolizing enzyme and is expressed in liver. Although human CYP2B6 variants account for variable enzyme properties among individuals and populations, CYP2B6 genetic variants have not been investigated in cynomolgus macaques, widely used in drug metabolism studies. CYP2B6 was resequenced in 120 cynomolgus macaques and 23 rhesus macaques by direct sequencing. Twenty-three non-synonymous variants were found, of which 12 and 3 were unique to cynomolgus macaques and rhesus macaques, respectively. By functional characterization using the 14 variant proteins, 8 variants (V114I, R253C, M435I, V459M, L465P, C475S, R487C, and R487H) showed different rate (>1.5-fold) of testosterone 16β-hydroxylation to wild type. However, the four variants (M435I, L465P, C475S, and R487H) were analyzed in liver microsomes, and the catalytic rates were not substantially different from wild type. Macaque CYP2B6 was polymorphic, and the genotype could partly account for variable enzyme activities of macaque CYP2B6. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rapid estimation of compost enzymatic activity by spectral analysis method combined with machine learning.

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Chakraborty, Somsubhra; Das, Bhabani S; Ali, Md Nasim; Li, Bin; Sarathjith, M C; Majumdar, K; Ray, D P

2014-03-01

The aim of this study was to investigate the feasibility of using visible near-infrared (VisNIR) diffuse reflectance spectroscopy (DRS) as an easy, inexpensive, and rapid method to predict compost enzymatic activity, which traditionally measured by fluorescein diacetate hydrolysis (FDA-HR) assay. Compost samples representative of five different compost facilities were scanned by DRS, and the raw reflectance spectra were preprocessed using seven spectral transformations for predicting compost FDA-HR with six multivariate algorithms. Although principal component analysis for all spectral pretreatments satisfactorily identified the clusters by compost types, it could not separate different FDA contents. Furthermore, the artificial neural network multilayer perceptron (residual prediction deviation=3.2, validation r(2)=0.91 and RMSE=13.38 μg g(-1) h(-1)) outperformed other multivariate models to capture the highly non-linear relationships between compost enzymatic activity and VisNIR reflectance spectra after Savitzky-Golay first derivative pretreatment. This work demonstrates the efficiency of VisNIR DRS for predicting compost enzymatic as well as microbial activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

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Avellaneda-Torres, Lizeth Manuela; León Sicard, Tomás Enrique; Torres Rojas, Esperanza

2018-08-01

significantly under potato cropping and livestock. The activities of β-glucosidase, acid phosphatase, alkaline phosphatase, phosphodiesterase and protease were higher during the dry season than the rainy season, and the activities of β-glucosidase, acid phosphatase and urease decreased statistically in the lower-altitude farm (La Secreta). These decreases in enzymatic activities are attributable to changes in the organic carbon of the soil. This study provides a novel insight on the relationships between land use and the physicochemical parameters and enzymatic activities of Páramo soils (which have been minimally studied to date) at different altitudes and during different seasons. The results suggest that changes in agricultural practices should be implemented to maintain the organic carbon of soil and, therefore, its enzymatic activities. Copyright © 2018 Elsevier B.V. All rights reserved.

cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

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Ushijima, Hironori [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan); Maeda, Masatomo, E-mail: mmaeda@iwate-med.ac.jp [Department of Molecular Biology, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba, Shiwagun, Iwate 028-3694 (Japan)

2012-07-13

Highlights: Black-Right-Pointing-Pointer A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6. Black-Right-Pointing-Pointer Effect of a JNK activator anisomycin on the proteolysis was examined. Black-Right-Pointing-Pointer Anisomycin stimulated the export of nuclear GATA-6 into the cytoplasm. Black-Right-Pointing-Pointer JNK activated the CRM1 mediated nuclear export of GATA-6. Black-Right-Pointing-Pointer JNK further stimulated slowly the degradation of GATA-6 by cytoplasmic proteasomes. -- Abstract: A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

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Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

2012-02-01

Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

International Nuclear Information System (INIS)

Ramanaviciene, A.; Kausaite-Minkstimiene, A.; Voronovic, J.; Ramanavicius, A.; Oztekin, Y.; Carac, G.; German, N.

2011-01-01

We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces. (author)

Enzymatic activity of anthropogenic proto-organic soils in soilless farming

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Bireescu, Geanina; Dazzi, Carmelo; Laudicina, Vito Armando; Lo Papa, Giuseppe

2017-04-01

In soilless agriculture and horticulture coir is the more used substratum to grow plants because it is widely available and more environmentally friendly than sphagnum or peat. In Italy, soilless agriculture concerns an area of about 1,000 hectares, particularly concentrated in Sicily. The southern coastal belt of this region is the area interested by the most significant experiences in the application of techniques of soilless cultivation that, recently, has been used also for growing table grapes. Starting from the above consideration we suppose that the features of the coconut fiber underlay an evident transformation and that even after few years of table grape cultivation, such organic material undergone to a transformation that allows for the formation of a proto-organic soil (a proto-Histosol, we supposed). If this is true, we believe that, in this case, to speak about soilless cultivation is for sure misleading for the common people, as we should define this cultivation "on anthropogenic soils" instead. To fit the aims of this survey we used a big greenhouse devoted to soilless cultivation of table grape in a farm in the Southern Sicily We have considered the enzymatic activity that characterized the coconut fiber after 3 cycles of cultivation of table grapes. We used as a control the coconut fiber that the farmer used to prepare pots for soilless cultivation and coconut fiber of: 6 pots at the end of the first productive cycle 6 pots at the end of the second cycle and 3 pots at the end of the third cycle. On these organic samples we investigated three enzymes, belonging to oxydoreductase (catalase and dehydrogenase) and hydrolase (urease) classes. Statistical analysis of the investigated enzymes was developed using IBM Statistic SPSS v20 by ANOVA, Tukey test HSD for p ≤ 0.01 and Multivariate Statistical Analysis. Results have shown significant differences in enzymes content and quality among coir tests. The use of the coco fiber, as nutritive substratum

Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

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Okba Selama

2014-01-01

Full Text Available Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

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Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

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SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

2016-01-01

SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

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Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

2015-12-21

Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

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Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

2013-09-05

The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Comparison of the role that entropy has played in processes of non-enzymatic and enzymatic catalysis

International Nuclear Information System (INIS)

Dixon Pineda, Manuel Tomas

2012-01-01

The function that entropy has played is compared in processes of non-enzymatic and enzymatic catalysis. The processes followed are showed: the kinetics of the acid hydrolysis of 3-pentyl acetate and cyclopentyl acetate catalyzed by hydrochloric acid and enzymatic hydrolysis of ethyl acetate and γ-butyrolactone catalyzed by pig liver esterase. The activation parameters of Eyring were determined for each process and interpreted the contribution of the entropy of activation for catalysis in this type of model reactions. (author) [es

Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

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Jindo, K.; Matsumoto, K.; García Izquierdo, C.; Sonoki, T.; Sanchez-Monedero, M. A.

2014-07-01

Biochar application has received increasing attention as a means to trap recalcitrant carbon and enhance soil fertility. Hydrolytic enzymatic assays, such as β-glucosidase and phosphatase activities, are used for the assessment of soil quality and composting process, which are based on use of p-nitrophenol (PNP) derivatives as substrate. However, sorption capacity of biochar can interfere with colorimetric determination of the hydrolysed PNP, either by the sorption of the substrate or the reaction product of hydrolysis into biochar surface. The aim of the present work is to study the biochar sorption capacity for PNP in biochar-blended composting mixtures in order to assess its impact on the estimation of the colorimetric-based enzymatic assays. A retention test was conducted by adding a solution of known amounts of PNP in universal buffer solution (pH = 5, 6.5 and 11, corresponding to the β-glucosidase, acid and alkaline phosphatase activity assays, respectively), in samples taken at the initial stage and after maturation stage from four different composting piles (two manure composting piles; PM: poultry manure, CM: cow manure and two other similar piles containing 10% of additional biochar (PM + B, CM + B)). The results show that biochar-blended composts (PM + B, CM + B) generally exhibited low enzymatic activities, compared to manure compost without biochar (PM, CM). In terms of the difference between the initial and maturation stage of composting process, the PNP retention in biochar was shown higher at maturation stage, caused most probably by an enlarged proportion of biochar inside compost mixture after the selective degradation of easily decomposable organic matter. TThe retention of PNP on biochar was influenced by pH dependency of sorption capacity of biochar and/or PNP solubility, since PNP was more efficiently retained by biochar at low pH values (5 and 6.5) than at high pH values (11).

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

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Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

Neutron diffraction studies on GdB6 and TbB6 powders

International Nuclear Information System (INIS)

Luca, S.E.; Amara, M.; Galera, R.M.; Givord, F.; Granovsky, S.; Isnard, O.; Beneu, B.

2004-01-01

We report here the first powder neutron diffraction study of GdB 6 and TbB 6 . GdB 6 and TbB 6 order antiferromagnetically at 15 and 21 K, respectively. In both compounds the transition at T N is of the first order. Moreover GdB 6 presents a second spontaneous magnetic transition at T * =8 K. The present study shows that, in both compounds, the magnetic propagation vectors belong to the star and that the direction of the magnetic moment is perpendicular to the ((1)/(2)) component of the wave-vector in GdB 6 , while in TbB 6 the moment is parallel to it. The deduced low-temperature values of the magnetic moments agree with those of the respective rare-earth trivalent ions

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs

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Ortega Ryan A

2011-02-01

Full Text Available Abstract Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs that sense enzymatic activity for applications in magnetic resonance imaging (MRI. To achieve this goal, we utilize amphiphilic poly(propylene sulfide-bl-poly(ethylene glycol (PPS-b-PEG copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

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Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

2011-02-27

Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative

Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids*

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Novellasdemunt, Laura; Tato, Irantzu; Navarro-Sabate, Aurea; Ruiz-Meana, Marisol; Méndez-Lucas, Andrés; Perales, Jose Carlos; Garcia-Dorado, David; Ventura, Francesc; Bartrons, Ramon; Rosa, Jose Luis

2013-01-01

Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked. PMID:23457334

Effect of MCM09, an active site-directed inhibitor of factor Xa, on B16-BL6 melanoma lung colonies in mice.

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Rossi, C; Hess, S; Eckl, R W; di Lena, A; Bruno, A; Thomas, O; Poggi, A

2006-03-01

Treatment with anticoagulant drugs has shown potential inhibitory effect on tumor invasion, although the relationship with clotting inhibition was not clear. The aim of our study was to evaluate the potential antitumor activity of MCM09, a newly developed, active site-directed, small molecule inhibitor of factor Xa (FXa) [WO0216312], and to relate the findings to anticlotting potency. MCM09 (0.1-10 mg kg(-1)) or heparin (H; 10 mg kg(-1)) was injected intravenously (i.v.), with 5 x 10(4) B16-BL6 melanoma cells, in C57BL/6 mice. Mice were killed after 18 days, to count lung colonies. Ex vivo anticoagulant activity was measured by activated partial thromboplastin time (APTT) on mouse plasma. MCM09, a selective inhibitor of FXa (IC-50 = 2.4 nm against human FXa), inhibited in a dose-dependent manner B16-BL6 melanoma lung colonies in mice. Mean lung metastasis number was 20.9 +/- 4.8 in controls (n = 10), 1.2 +/- 0.4 in mice treated with H, 10 mg kg(-1) i.v. (P < 0.01), 0.9 +/- 0.3, 9.2 +/- 2.2 and 15.5 +/- 2.6 in mice treated with MCM09, at 10 (P < 0.01), 1 (P < 0.05) and 0.1 mg kg(-1) i.v. (ns), respectively. MCM09 (10 mg kg(-1) i.v.) significantly prolonged APTT (57.1 +/- 10.2 s) 30 min after i.v. injection when compared with controls (25.3 +/- 1.6 s; P < 0.05). Lung colonies were 74.2-72.6% reduced by MCM09 (10 mg kg(-1)) given 60 or 120 min before cells, but not by MCM09 given 60 min thereafter, suggesting a direct cell interaction as a mechanism underlying antitumor activity.

Optimization of enzymatic clarification of green asparagus juice using response surface methodology.

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Chen, Xuehong; Xu, Feng; Qin, Weidong; Ma, Lihua; Zheng, Yonghua

2012-06-01

Enzymatic clarification conditions for green asparagus juice were optimized by using response surface methodology (RSM). The asparagus juice was treated with pectinase at different temperatures (35 °C-45 °C), pH values (4.00-5.00), and enzyme concentrations (0.6-1.8 v/v%). The effects of enzymatic treatment on juice clarity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity were investigated by employing a 3-factor central composite design coupled with RSM. According to response surface analysis, the optimal enzymatic treatment condition was pectinase concentration of 1.45%, incubation temperature of 40.56 °C and pH of 4.43. The clarity, juice yield, and soluble solid contents in asparagus juice were significantly increased by enzymatic treatment at the optimal conditions. DPPH radical-scavenging capacity was maintained at the level close to that of raw asparagus juice. These results indicated that enzymatic treatment could be a useful technique for producing green asparagus juice with high clarity and high-antioxidant activity. Treatment with 1.45% pectinase at 40.56 ° C, pH 4.43, significantly increased the clarity and yield of asparagus juice. In addition, enzymatic treatment maintained antioxidant activity. Thus, enzymatic treatment has the potential for industrial asparagus juice clarification. © 2012 Institute of Food Technologists®

Assessment of Napropamide Dissipation and its Effect on Soil Enzymatic Activity

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Mirosław Onyszko

2017-11-01

Full Text Available This paper assesses the dissipation of napropamide and its impact on the activity of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease in sandy clay loam. The experiment was carried out on soil samples with organic carbon content of 12.08 g·kg-1, total nitrogen content of 0.97 g·kg-1, and pH 5.24 with the following variable factors: (a dose of Devrinol 450 SC formation (containing 450 g of napropamide in dm3: 0 (control, 0.5, 1, 2, 4, 8, and 16-fold hold of field dose; (b day of experiment: 1, 7, 14, 28, 56, and 112. The half-life of napropamide ranged from 33.50 to 71.42 days. The use of napropamide at the dose recommended by the manufacturer and at the dose reduced by half appeared to exhibit low toxicity in relation to enzymes determined. In contrast, the application of elevated napropamide doses decreased the values of biochemical parameters of the soil in most cases. The Pearson correlation coefficients showed statistically significant negative correlation between the content of napropamide residues and the enzymatic activity of the soil.

Sangivamycin-Like Molecule 6 (SLM6) exhibits potent anti-multiple myeloma activity through inhibition of cyclin-dependent kinase-9 (CDK9)

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Dolloff, Nathan G.; Allen, Joshua E.; Dicker, David T.; Aqui, Nicole; Vogl, Dan; Malysz, Jozef; Talamo, Giampaolo; El-Deiry, Wafik S.

2012-01-01

Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLMs). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or non-malignant cell lines at sub-micromolar concentrations. SLM6 was the most active compound in vivo, where it was well-tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (c-Maf, cyclin D1, and c-Myc). Furthermore, SLM6 demonstrated superior in vivo anti-MM activity over the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings demonstrate that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent. PMID:22964485

Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

LENUS (Irish Health Repository)

Killeen, S D

2009-05-19

Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

Decreased enzymatic activity of 5,10-methylene tetrahydrofolate reductase affects the development of several diseases

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Maša Vidmar

2016-07-01

Full Text Available The importance of folates in human physiology is well known, as are various pathologies associated with low folate status. Folate deficiency can occur due to low dietary intake, genetic predisposition or treatment with medicines affecting the folate status. The aim of this paper is to explore the importance of determining genetic polymorphisms which influence the levels of biologically active folate. MTHFR is involved in the transformation of 5,10-methylene-THF to 5-methyl-THF. Polymorphisms of the MTHRF gene are associated with decreased enzymatic activity.Only 9.3 % of the population in Slovenia displays full activity of the MTHFR enzyme; these subjects are non-mutated homozygotes (wild-type alleles. In contrast, the average enzymatic activity in subjects with mutated alleles is between 50 and 60 %. MTHFR polymorphism is associated with an increased risk of hyperhomocysteinemia and cardiovascular diseases, neurological disorders and various types of cancer. There is also an increased risk for congenital malformations. Folic acid food fortification was introduced in some countries in order to assure an adequate folate status in the population. However, this approach does not address the decreased activity of MTHFR.Polymorphism in the key enzymes of the folate cycle is common. Determination of the genetic predisposition is therefore plausible in the most vulnerable groups of the population, such as pregnant women and patients receiving medicines influencing the folate cycle in various ways, e.g. 5-fluorouracil, methotrexate and 6-mercaptopurine. Genotyping would allow the identification of patients at high risk for suboptimal folate status.

ASPECTS CONCERNING THE ENZYMATIC ACTIVITY IN SEVERAL THERMOACTINOMYCETE STRAINS

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Simona Dunca

2003-08-01

Full Text Available In the thermoactinomycete strains subjected to examination the values of their recorded enzymatic activities (i.e. α-amy lase, protease, exo-β-1,4 – glucanase, endo -β-1,4 – glucanase and β-glucosidase were lower in the stationary cultures as compared to the stirred ones. The strain Thermomonospora fusca BB255 was found to be highly cellulase- producing and at the same time able to synthesize α-amy lases and proteases.

Improvement of biological nitrogen removal with nitrate-dependent Fe(II) oxidation bacterium Aquabacterium parvum B6 in an up-flow bioreactor for wastewater treatment.

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Zhang, Xiaoxin; Li, Ang; Szewzyk, Ulrich; Ma, Fang

2016-11-01

Aquabacterium parvum strain B6 exhibited efficient nitrate-dependent Fe(II) oxidation ability using nitrate as an electron acceptor. A continuous up-flow bioreactor that included an aerobic and an anoxic section was constructed, and strain B6 was added to the bioreactor as inocula to explore the application of microbial nitrate-dependent Fe(II) oxidizing (NDFO) efficiency in wastewater treatment. The maximum NRE (anoxic section) and TNRE of 46.9% and 79.7%, respectively, could be obtained at a C/N ratio of 5.3:1 in the influent with HRT of 17. Meanwhile, the taxonomy composition of the reactor was assessed, as well. The NDFO metabolism of strain B6 could be expected because of its relatively dominant position in the anoxic section, whereas potential heterotrophic nitrification and aerobic denitrification developed into the prevailing status in the aerobic section after 50days of continuous operation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis

Energy Technology Data Exchange (ETDEWEB)

Bouetard, Anthony, E-mail: anthony.bouetard@rennes.inra.fr [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France); Besnard, Anne-Laure; Vassaux, Daniele; Lagadic, Laurent; Coutellec, Marie-Agnes [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France)

2013-01-15

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 {mu}g l{sup -1}) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

PEG-b-PCL polymeric nano-micelle inhibits vascular angiogenesis by activating p53-dependent apoptosis in zebrafish.

Science.gov (United States)

Zhou, Tian; Dong, Qinglei; Shen, Yang; Wu, Wei; Wu, Haide; Luo, Xianglin; Liao, Xiaoling; Wang, Guixue

Micro/nanoparticles could cause adverse effects on cardiovascular system and increase the risk for cardiovascular disease-related events. Nanoparticles prepared from poly(ethylene glycol) (PEG)- b -poly( ε -caprolactone) (PCL), namely PEG- b -PCL, a widely studied biodegradable copolymer, are promising carriers for the drug delivery systems. However, it is unknown whether polymeric PEG- b -PCL nano-micelles give rise to potential complications of the cardiovascular system. Zebrafish were used as an in vivo model to evaluate the effects of PEG- b -PCL nano-micelle on cardiovascular development. The results showed that PEG- b -PCL nano-micelle caused embryo mortality as well as embryonic and larval malformations in a dose-dependent manner. To determine PEG- b -PCL nano-micelle effects on embryonic angiogenesis, a critical process in zebrafish cardiovascular development, growth of intersegmental vessels (ISVs) and caudal vessels (CVs) in flk1-GFP transgenic zebrafish embryos using fluorescent stereomicroscopy were examined. The expression of fetal liver kinase 1 (flk1), an angiogenic factor, by real-time quantitative polymerase chain reaction (qPCR) and in situ whole-mount hybridization were also analyzed. PEG- b -PCL nano-micelle decreased growth of ISVs and CVs, as well as reduced flk1 expression in a concentration-dependent manner. Parallel to the inhibitory effects on angiogenesis, PEG- b -PCL nano-micelle exposure upregulated p53 pro-apoptotic pathway and induced cellular apoptosis in angiogenic regions by qPCR and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay. This study further showed that inhibiting p53 activity, either by pharmacological inhibitor or RNA interference, could abrogate the apoptosis and angiogenic defects caused by PEG- b -PCL nano-micelles, indicating that PEG- b -PCL nano-micelle inhibits angiogenesis by activating p53-mediated apoptosis. This study indicates that polymeric PEG- b -PCL nano-micelle could

Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting.

Science.gov (United States)

Nikaeen, Mahnaz; Nafez, Amir Hossein; Bina, Bijan; Nabavi, BiBi Fatemeh; Hassanzadeh, Akbar

2015-05-01

The objective of this work was to study the evolution of physico-chemical and microbial parameters in the composting process of sewage sludge (SS) with pruning wastes (PW) in order to compare these parameters with respect to their applicability in the evaluation of organic matter (OM) stabilization. To evaluate the composting process and organic matter stability, different microbial activities were compared during composting of anaerobically digested SS with two volumetric ratios, 1:1 and 3:1 of PW:SS and two aeration techniques including aerated static piles (ASP) and turned windrows (TW). Dehydrogenase activity, fluorescein diacetate hydrolysis, and specific oxygen uptake rate (SOUR) were used as microbial activity indices. These indices were compared with traditional parameters, including temperature, pH, moisture content, organic matter, and C/N ratio. The results showed that the TW method and 3:1 (PW:SS) proportion was superior to the ASP method and 1:1 proportion, since the former accelerate the composting process by catalyzing the OM stabilization. Enzymatic activities and SOUR, which reflect microbial activity, correlated well with temperature fluctuations. Based on these results it appears that SOUR and the enzymatic activities are useful parameters to monitor the stabilization of SS compost. Copyright © 2015 Elsevier Ltd. All rights reserved.

25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

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Ichikawa, Tomohiro [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Sugiura, Hisatoshi, E-mail: sugiura@rm.med.tohoku.ac.jp [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan); Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Ichinose, Masakazu [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan)

2013-05-01

Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblasts (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P<0.001) and collagen I (P<0.001) expression in HFL-1. 25-HC also significantly enhanced the release and activation of matrix metallaoproteinase (MMP)-2 (P<0.001) and MMP-9 (P<0.001) without any significant effect on the production of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. 25-HC stimulated transforming growth factor (TGF)-β{sub 1} production (P<0.01) and a neutralizing anti-TGF-β antibody restored these 25-HC-augmented pro-fibrotic responses. 25-HC significantly promoted the translocation of nuclear factor (NF)-κB p65 into the nuclei (P<0.01), but not phospholylated-c-jun, a complex of activator protein-1. Pharmacological inhibition of NF-κB restored the 25-HC-augmented pro-fibrotic responses and TGF-β{sub 1} release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Science.gov (United States)

Thakur, Manoj; Kumar, Mohan B J; Muniyappa, K

2016-10-18

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome.

Science.gov (United States)

Hoekstra, Elmer; Das, Asha M; Swets, Marloes; Cao, Wanlu; van der Woude, C Janneke; Bruno, Marco J; Peppelenbosch, Maikel P; Kuppen, Peter J K; Ten Hagen, Timo L M; Fuhler, Gwenny M

2016-04-19

Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.

Lactose hydrolysis in an enzymatic membrane reactor

Energy Technology Data Exchange (ETDEWEB)

Mertens, B; Huyghebaert, A

1987-10-01

The enzymatic hydrolysis of lactose in whey permeate with subsequent recuperation of Saccharomyces lactis lactase by means of ultrafiltration was investigated. In whey permeate, S. lactis lactase shows maximal activity at pH 6.5; the optimal temperature was found to be 45/sup 0/C and is limited by strong thermal inactivation beyond this temperature. High activity combined with acceptable thermal inactivation (< 10% after 5 h incubation) was established at 30/sup 0/C. S. lactis lactase also displays considerable activity at low temperature (5/sup 0/C). Enzyme stability is reduced drastically by demineralisation: addition of low concentrations of manganese ions (10/sup -3/ M) considerably enhances stability. Using a DDS Lab-Unit 35 fitted with GR61PP polysulphon membranes (cut-off: 20.000), pilot scale experiments were carried out (pH 6.5; 30/sup 0/C) in which whey permeate was hydrolyzed to a degree of hydrolysis of 82% minimum. Enzyme recuperation amounted to 96.5% per batch, all enzyme activity loss being due to thermal inactivation. Microbiological examination of the enzymatic membrane reactor showed that growth of mcicroorganisms can largely be suppressed by working at lower temperature (5/sup 0/C). Eventually, 50 ppm H/sub 2/O/sub 2/ or sterile filtration will adequately solve microbiological problems without affecting enzyme activity.

BnaA.bZIP1 Negatively Regulates a Novel Small Peptide Gene, BnaC.SP6, Involved in Pollen Activity

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Xuanpeng Wang

2017-12-01

Full Text Available Small peptides secreted to the extracellular matrix control many aspects of the plant’s physiological activities which were identified in Arabidopsis thaliana, called ATSPs. Here, we isolated and characterized the small peptide gene Bna.SP6 from Brassica napus. The BnaC.SP6 promoter was cloned and identified. Promoter deletion analysis suggested that the -447 to -375 and -210 to -135 regions are crucial for the silique septum and pollen expression of BnaC.SP6, respectively. Furthermore, the minimal promoter region of p158 (-210 to -52 was sufficient for driving gene expression specifically in pollen and highly conserved in Brassica species. In addition, BnaA.bZIP1 was predominantly expressed in anthers where BnaC.SP6 was also expressed, and was localized to the nuclei. BnaA.bZIP1 possessed transcriptional activation activity in yeast and protoplast system. It could specifically bind to the C-box in p158 in vitro, and negatively regulate p158 activity in vivo. BnaA.bZIP1 functions as a transcriptional repressor of BnaC.SP6 in pollen activity. These results provide novel insight into the transcriptional regulation of BnaC.SP6 in pollen activity and the pollen/anther-specific promoter regions of BnaC.SP6 may have their potential agricultural application for new male sterility line generation.

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

International Nuclear Information System (INIS)

Jeng, Yow-Jiun; Watson, Cheryl S

2009-01-01

Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH 3 /B 6 /F 10 , originally subcloned from GH 3 cells based on its ability to express high levels of the membrane estrogen receptor-α. We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH 3 /B 6 /F 10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary

Porcine arterivirus activates the NF-κB pathway through IκB degradation

International Nuclear Information System (INIS)

Lee, Sang-Myeong; Kleiboeker, Steven B.

2005-01-01

Nuclear factor-kappaB (NF-κB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-κB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-κB activation was characterized by translocation of NF-κB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-κB-regulated gene expression. NF-κB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-κB activation. Degradation of IκB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IκBα (IκBαDN) significantly suppressed NF-κB activation induced by PRRSV. However, IκBαDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-κB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-κB was activated by PRRSV infection. Moreover, NF-κB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-κB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV

Some Anilides of 2-Alkylthio- and 2-Chloro-6-Alkylthio-4-Pyridinecarboxylic Acids: Synthesis and Photosynthesis-Inhibiting Activity

Directory of Open Access Journals (Sweden)

Miloš Macháček

2001-06-01

Full Text Available Many compounds containing a -CONH- group display photosynthesis inhibiting activity. Based on this structural feature, a group of anilides of 2-alkylthio-(1b-4f or 2-chloro-6-alkylthio-4-pyridinecarboxylic acids (5a-6c was synthesised. The prepared compounds were tested for their inhibition of the oxygen evolution rate (OER in spinach chloroplasts. A quasi-parabolic dependence between photosynthesis-inhibiting activity and the lipophilicity of the compounds was determined for 1b-4f as well as for 5a-6c. The inhibitory activity of compounds 1b-4f was higher than that of 5a-6c for comparable lipophilicity values.

Monoamine oxidase B (MAO-B) inhibition by active principles from Uncaria rhynchophylla.

Science.gov (United States)

Hou, Wen-Chi; Lin, Rong-Dih; Chen, Cheng-Tang; Lee, Mei-Hsien

2005-08-22

Attenuation of monoamine oxidase B (MAO-B) activity may provide protection against oxidative neurodegeneration. For this reason, inhibition of MAO-B activity is used as part of the treatment of Parkinson's and Alzheimer's patients. The hook of Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) is a traditional Chinese herbal drug that is generally used to treat convulsive disorders. In this study, the fractionation and purification of Uncaria rhynchophylla extracts using a bioguided assay isolated two known compounds, (+)-catechin and (-)-epicatechin. The compounds inhibited MAO-B, as measured by an assay of rat brain MAO-B separated by electrophoresis on a 7.5% native polyacrylamide gel. The IC(50) values of (+)-catechin and (-)-epicatechin were 88.6 and 58.9 microM, respectively, and inhibition occurred in a dose-dependent manner, as measured by the fluorescence method. The Lineweaver-Burk plot revealed K(i) values for (+)-catechin and (-)-epicatechin of 74 and 21 microM, respectively. This suggests that these two compounds, isolated here for the first time from Uncaria rhynchophylla, might be able to protect against neurodegeneration in vitro, and, therefore, the molecular mechanism deserves further study. This finding may also increase interest in the health benefits of Uncaria rhynchophylla.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the third isozyme with a distinct expression profile.

Science.gov (United States)

Nomura, Taiji; Kuchida, Ryo; Kitaoka, Naoki; Kato, Yasuo

2018-02-23

6-Tuliposide B (PosB), a major secondary metabolite that accumulates in tulip (Tulipa gesneriana), is converted to the antibacterial lactone, tulipalin B (PaB), by PosB-converting enzyme (TCEB). TgTCEB1 and TgTCEB-R, which encode TCEB, are specifically expressed in tulip pollen and roots, respectively, but are hardly expressed in other tissues (e.g. leaves) despite the presence of substantial PosB-converting activity, suggesting the existence of another TCEB isozyme. Here, we describe the identification of TgTCEB-L ("L" for leaf), a paralog of TgTCEB1 and TgTCEB-R, from leaves via native enzyme purification. The enzymatic characters of TgTCEB-L, including catalytic activity and subcellular localization, were substantially the same as those of TgTCEB1 and TgTCEB-R. However, TgTCEB-L did not exhibit tissue-specific expression. Identification of TgTCEB-L explains the PosB-converting activity detected in tissues where TgTCEB1 and TgTCEB-R transcripts could not be detected, indicating that tulip subtilizes the three TgTCEB isozymes depending on the tissue.

Temperature dependence of the activity of polyphenol peroxidases and polyphenol oxidases in modern and buried soils

Science.gov (United States)

Yakushev, A. V.; Kuznetsova, I. N.; Blagodatskaya, E. V.; Blagodatsky, S. A.

2014-05-01

Under conditions of the global climate warming, the changes in the reserves of soil humus depend on the temperature sensitivities of polyphenol peroxidases (PPPOs) and polyphenol oxidases (PPOs). They play an important role in lignin decomposition, mineralization, and humus formation. The temperature dependence of the potential enzyme activity in modern and buried soils has been studied during incubation at 10 or 20°C. The experimental results indicate that it depends on the availability of the substrate and the presence of oxygen. The activity of PPOs during incubation in the absence of oxygen for two months decreases by 2-2.5 times, which is balanced by an increase in the activity of PPPOs by 2-3 times. The increase in the incubation temperature to 20°C and the addition of glucose accelerates this transition due to the more abrupt decrease in the activity of PPOs. The preincubation of the soil with glucose doubles the activity of PPPOs but has no significant effect on the activity of PPOs. The different effects of temperature on two groups of the studied oxidases and the possibility of substituting enzymes by those of another type under changing aeration conditions should be taken into consideration in predicting the effect of the climate warming on the mineralization of the soil organic matter. The absence of statistically significant differences in the enzymatic activity between the buried and modern soil horizons indicates the retention by the buried soil of some of its properties (soil memory) and the rapid restoration of high enzymatic activity during the preincubation.

Mechano-Enzymatic Deconstruction with a New Enzymatic Cocktail to Enhance Enzymatic Hydrolysis and Bioethanol Fermentation of Two Macroalgae Species

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Sameh Amamou

2018-01-01

Full Text Available The aim of this study was to explore the efficiency of a mechano-enzymatic deconstruction of two macroalgae species for sugars and bioethanol production, by using a new enzymatic cocktail (Haliatase and two types of milling modes (vibro-ball: VBM and centrifugal milling: CM. By increasing the enzymatic concentration from 3.4 to 30 g/L, the total sugars released after 72 h of hydrolysis increased (from 6.7 to 13.1 g/100 g TS and from 7.95 to 10.8 g/100 g TS for the green algae U. lactuca and the red algae G. sesquipedale, respectively. Conversely, total sugars released from G. sesquipedale increased (up to 126% and 129% after VBM and CM, respectively. The best bioethanol yield (6 geth/100 g TS was reached after 72 h of fermentation of U. lactuca and no increase was obtained after centrifugal milling. The latter led to an enhancement of the ethanol yield of G. sesquipedale (from 2 to 4 g/100 g TS.

Enzymatic reduction of U(VI) in groundwaters

International Nuclear Information System (INIS)

Addelouas, A.; Gong, W.; Lutze, W.; Nuttall, E.; Fritz, B.; Crovisier, J.L.

1999-01-01

The use of enzymatic reduction of U(VI) in remediation of groundwater contaminated with U(VI) is receiving considerable attention. Certain strains of bacteria can combine the oxidation of an organic compound to the reduction of U(VI) to U(IV), which precipitates as uraninite. In the present study, we tested the reduction of U(VI) in groundwaters with various origins and compositions. In all groundwaters u(VI) was reduced by sulfate reducing bacteria that had been activated by ethanol and tri-metaphosphate. The reduction rate of U(VI) depends on sulfate concentration in water and the abundance of bacteria in the system. This work shows that bacteria capable of U(VI) reduction are ubiquitous in nature, and suggests the possibility of a large application of the enzymatic reduction of U(VI) for in situ clean up of groundwaters contaminated with uranium. (authors)

Ordering and Fluctuation of Quantum Multipoles in CeB6

International Nuclear Information System (INIS)

Shiina, R.

2003-01-01

The effect of multipolar fluctuations on the quadrupolar phase transition in CeB 6 is investigated theoretically. It is shown that the fluctuations become strong and field-dependent, reflecting the competition of coupled multipolar interactions. Some unusual phenomena around the transition in CeB 6 are shown to be reasonably explained within the RKKY model. (author)

ASSOCIATION BETWEEN ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE

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A. Vaisi-Raygani

2008-04-01

Full Text Available The etiopathogenesis of dementia in Alzheimer's disease (AD is still unclear. However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111±324 U/grHb, 43.7±11.6 U/grHb, and 1.17 ±0.23 mmol/L compared with 1371±211 U/gHb; t= -2.17, p=0.036, 56.3±9.5 U/gHb; t=3.8, p=0.014, and 1.54±0.2 mmol/L; t=11.18, P<0.001, respectively. While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t=1.3, P=0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD.

ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE1

Directory of Open Access Journals (Sweden)

A. Vaisi-Raygani

2007-07-01

Full Text Available The etiopathogenesis of Alzheimer's disease (AD is still unclear.  However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111 ± 324 U/grHb, 43.7 ± 11.6 U/grHb, and 1.17 ± 0.23 mmol/l compared with 1371 ± 211 U/grHb; t= -2.17, P = 0.036, 56.3 ± 9.5 U/grHb; t=3.8, P = 0.014, and 1.54±0.2 mmol/l; t=11.18, P < 0.001, respectively.  While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t = 1.3, P = 0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD. 

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

International Nuclear Information System (INIS)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

New Biocatalyst with Multiple Enzymatic Activities for Treatment of Complex Food Wastewaters

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Olga Senko

2008-01-01

Full Text Available The cells of filamentous fungus R. oryzae entrapped in the polyvinyl alcohol cryogelare capable of producing various extracellular hydrolytic enzymes (proteases, amylases, lipases and are used for the treatment of complex wastewaters of food industry. Five types of media simulating the wastewater of various food enterprises were treated under batch conditions for 600 h. Fats containing mostly residues of unsaturated fatty acids, as well as casein, glucose, sucrose, starch, soybean flour and various salts were the main components of the treated wastewaters. The immobilized cells concurrently possessed lipolytic, amylolytic and proteolytic activities. The level of each enzymatic activity depended on the wastewater content. The physiological state of immobilized cells was monitored by bioluminescent method. The intracellular adenosine triphosphate (ATP concentration determined in the granules with immobilized cells was high enough and almost constant for all the period of biocatalyst application confirming thereby the active metabolic state of the cells. The study of mechanical strength of biocatalyst granules allowed revealing the differences in the values of modulus of biocatalyst elasticity at the beginning and at the end of its use for the wastewater treatment. The decrease in chemical oxygen demand of the tested media after their processing by immobilized biocatalyst was 68–79 % for one working cycle.

MexiB 6 AS A RESULT OF FORTIFICATION OF ETHYLMETHYLHYDROXYPYRIDINE SUCCINATE WITH MAGNESIUM AND PYRIDOXINE: PROTEOME EFFECTS

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O. A. Gromova

2016-01-01

Full Text Available The efficacy of ethylmethylhydroxypyridine succinate (EMHPS  depends on the concentration and activity of proteomic proteins. To provide the body with magnesium and pyridoxine is an important condition for the efficacy of EMHPS because these micronutrients are essential for maintaining neuronal function.Objective: to analyze the biological effects of pyridoxineand magnesium-dependent proteins providing the molecular mechanisms of multi-targeted action of EMHPS,  pyridoxine, and magnesium.Material and methods. Proteins that interact with both pyridoxine and magnesium were found in the genomic and proteomic databases. A list of 78 vitamin B6-dependent proteins, which is based on the available human genome records in NCBIPROTEIN, EMBL,  UNIPROT,  and HumanProteomeMap databases, was analyzed using a functional binding assay. The same assay was also applied to analyze a list 720 magnesium-dependent proteins.Results. The analysis has shown that 78 pyridoxine-dependent proteomic proteins are necessary for: 1 the synthesis and processing of amino acids; 2 cell energy metabolism (ATP synthesis, and 3 the synthesis of neurotransmitters and neuronal membranes. MexiB 6 has numerous synergistic interactions between the molecules of EMHPS,  pyridoxine, and magnesium. The combination of the components of EMHPS,  pyridoxine and magnesium in MexiB 6 (triple synergism allows prediction that the drug has more pronounced clinical effects than the molecules of EMHPS,  which emerges in its antihypoxic and antioxidant activities, the improvement of synaptic transmission of a signal, the neutralization of homocysteine, and the regulation of lipid and carbohydrate metabolism (restoration of cell sensitivity to insulin and carbohydrates in patients with atherosclerosis and in those at risk for diabetes mellitus or obesity. Pyridoxineand magnesium-induced potentiation of the effects of EMHPS may enhance its activity.

Different Levels of Skin Whitening Activity among 3,6-Anhydro-l-galactose, Agarooligosaccharides, and Neoagarooligosaccharides

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Ji Hye Kim

2017-10-01

Full Text Available 3,6-Anhydro-l-galactose (AHG, a major monomeric constituent of red macroalgae (Rhodophyta, was recently reported to possess skin whitening activity. Moreover, AHG-containing oligosaccharides, such as agarooligosaccharides (AOSs and neoagarooligosaccharides (NAOSs, have various physiological activities, including anti-inflammatory, antioxidant, and skin moisturizing effects. In this study, AHG and NAOSs were produced from agarose by enzymatic reactions catalyzed by an endo-type β-agarase, an exo-type β-agarase, and a neoagarobiose hydrolase. In a cell proliferation assay, AHG, AOSs, and NAOSs at 12.5, 25, and 50 μg/mL concentrations did not exhibit cytotoxicity toward murine B16 melanoma cells or human epidermal melanocytes. In an in vitro skin whitening activity assay of AHG, AOSs, and NAOSs at 50 μg/mL, AHG showed the highest skin whitening activity in both murine B16 melanoma cells and human epidermal melanocytes; this activity was mediated by the inhibition of melanogenesis. Neoagarotetraose and neoagarohexaose also exhibited in vitro skin whitening activity, whereas neoagarobiose and AOSs with degrees of polymerization of 3 (agarotriose, 5 (agaropentaose, and 7 (agaroheptaose did not. Therefore, AHG is responsible for the skin whitening activity of agar-derived sugars, and the structural differences among the AHG-containing oligosaccharides may be responsible for their different skin whitening activities.

Electronic, thermodynamics and mechanical properties of LaB6 from first-principles

Science.gov (United States)

Ivashchenko, V. I.; Turchi, P. E. A.; Shevchenko, V. I.; Medukh, N. R.; Leszczynski, Jerzy; Gorb, Leonid

2018-02-01

Up to date, the electronic structure properties of amorphous lanthanum hexaboride, a-LaB6, were not yet investigated, and the thermodynamic and mechanical properties of crystalline lanthanum hexaboride (c-LaB6) were studied incompletely. The goal of this work was to fill these gaps in the study of lanthanum hexaborides. The electronic and phonon structures, thermodynamic and mechanical properties of both crystalline and amorphous lanthanum hexaborides (c-LaB6, a-LaB6, respectively) were investigated within the density functional theory. An amorphyzation of c-LaB6 gives rise to the metal - semiconductor transition. The thermal conductivity decreases on going from c-LaB6 to a-LaB6. The elastic moduli, hardness, ideal tensile and shear strengths of a-LaB6 are significantly lower compared to those of the crystalline counterpart, despite the formation of the icosahedron-like boron network in the amorphous phase. For c-LaB6, the stable boron octahedrons are preserved after the failure under tensile and shear strains. The peculiarity in the temperature dependence of heat capacity, Cp(T), at 50 K is explained by the availability of a sharp peak at 100 cm-1 in the phonon density of states of c-LaB6. An analysis of the Fermi surface indicates that this peak is not related to the shape of the Fermi surface, and is caused by the vibration of lanthanum atoms. In the phonon spectrum of a-LaB6, the peak at 100 cm-1 is significantly broader than in the spectrum of c-LaB6, for which reason the anomaly in the Cp(T) dependence of a-LaB6 does not appear. The calculated characteristics are in good agreement with the available experimental data.

Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner

DEFF Research Database (Denmark)

Kofod-Olsen, Emil; Ross-Hansen, Katrine; Mikkelsen, Jacob Giehm

2008-01-01

Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17-U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19...

[HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

Science.gov (United States)

Xu, Yan; Cheng, Hao; Zhao, Ke-Jia; Zhu, Ke-Jian; Zhang, Xing

2007-11-01

This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.

Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.

Science.gov (United States)

Keogh, Ciara E; Scholz, Carsten C; Rodriguez, Javier; Selfridge, Andrew C; von Kriegsheim, Alexander; Cummins, Eoin P

2017-07-07

CO 2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO 2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO 2 are not fully elucidated. Here, we identify several novel CO 2 -dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO 2 A cohort of RelB protein-protein interactions ( e.g. with Raf-1 and IκBα) are altered by CO 2 exposure, although others are maintained ( e.g. with p100). RelB is processed by CO 2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO 2 Thus, we provide molecular insight into the CO 2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO 2 -dependent regulation of inflammatory signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

Science.gov (United States)

Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

1992-01-01

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway.

Science.gov (United States)

Im, Eun Ju; Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu; Rhee, Man Hee

2016-01-01

Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway.

Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

Science.gov (United States)

Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

2017-04-01

Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems

DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

DEFF Research Database (Denmark)

Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust

2013-01-01

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I....... The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor....... The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented...

ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

DEFF Research Database (Denmark)

Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel

2012-01-01

Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function...... as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified...

Restriction of human herpesvirus 6B replication by p53

DEFF Research Database (Denmark)

Øster, Bodil; Kofod-Olsen, Emil; Bundgaard, Bettina

2008-01-01

Human herpesvirus 6B (HHV-6B) induces significant accumulation of p53 in both the nucleus and cytoplasm during infection. Activation of p53 by DNA damage is known to induce either growth arrest or apoptosis; nevertheless, HHV-6B-infected cells are arrested in their cell cycle independently of p53...

Solid-liquid stable phase equilibria of the ternary systems MgCl2 + MgB6O10+ H2O AND MgSO4 + MgB6O10 + H2O at 308.15 K

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Lingzong Meng

2014-03-01

Full Text Available The solubilities and the relevant physicochemical properties of the ternary systems MgCl2 + MgB6O10 + H2O and MgSO4 + MgB6O10 + H2O at 308.15 K were investigated using an isothermal dissolution method. It was found that there is one invariant point, two univariant curves, and two crystallization regions of the systems. The systems belong to a simple co-saturated type, and neither double salts nor solid solutions were found. Based on the extended HW model and its temperature-dependent equations, the single-salt Pitzer parameters β(0, β(1, β(2 and CØ for MgCl2, MgSO4, and Mg(B6O7(OH6, the mixed ion-interaction parameters θCl,B6O10, θSO4,B6O10, ΨMg,Cl,B6O10, ΨMg,SO4,B6O10 of the systems at 308.15 K were fitted, In addition, the average equilibrium constants of the stable equilibrium solids at 308.15 K were obtained by a method using the activity product constant. Then the solubilities of the ternary systems are calculated. The calculated solubilities agree well with the experimental values.

Human Papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

Science.gov (United States)

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

2012-01-01

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone. PMID:22284893

2',4-Dihydroxy-3',4',6'-trimethoxychalcone from Chromolaena odorata possesses anti-inflammatory effects via inhibition of NF-κB and p38 MAPK in lipopolysaccharide-activated RAW 264.7 macrophages.

Science.gov (United States)

Dhar, Rana; Kimseng, Rungruedee; Chokchaisiri, Ratchanaporn; Hiransai, Poonsit; Utaipan, Tanyarath; Suksamrarn, Apichart; Chunglok, Warangkana

2018-02-01

Immune dysregulation has been implicated in the pathogenesis of many diseases. Macrophages play a crucial role contributing to the onset, progression, and resolution of inflammation. Macrophage inflammatory mediators are of considerable interest as potential targets to treat inflammatory diseases. The present study was conducted to elucidate the anti-inflammatory mechanism of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1), the major chalcone isolated from Chromolaena odorata (L.) R.M.King & H.Rob, against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. Cell viability, nitric oxide (NO), and proinflammatory cytokines of LPS-activated RAW 264.7 cells were measured by MTT, Griess, and ELISA assays, respectively. Cell lysates were subjected to Western blotting for investigation of protein expression. Treatment with the major chalcone 1 significantly attenuated the production of NO and proinflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in a dose-dependent manner. The chalcone suppressed nuclear factor-κB (NF-κB) stimulation by preventing activation of inhibitor κB kinase (IKK) α/β, degradation of inhibitor κB (IκB) α, and translocation of p65 NF-κB into the nucleus. Additionally, the chalcone markedly repressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but no further inhibition was detected for c-Jun N-terminal activated kinases or extracellular regulated kinases. Thus, suppression of NF-κB and p38 MAPK activation may be the core mechanism underlying the anti-inflammatory activity of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1). These findings provide evidence that 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1) possesses anti-inflammatory activity via targeting proinflammatory macrophages. This anti-inflammatory chalcone is a promising compound for reducing inflammation.

Vitamin B6

Science.gov (United States)

... pork Nuts Poultry Whole grains and fortified cereals Canned chickpeas Fortified breads and cereals may also contain vitamin B6. Fortified means that a vitamin or mineral has been added to the food. Side Effects Large doses of vitamin B6 can cause: Difficulty ...

The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

Science.gov (United States)

Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

2016-07-01

In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m -2 ) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m -2 dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m -2 produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

Science.gov (United States)

Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

2017-06-01

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Supply with the vitamins B1, B2 and B6 in carcinomas before and after radiotherapy

International Nuclear Information System (INIS)

Grimm, U.; Wulff, K.; Schmidt, W.

1983-01-01

In 108 breast cancer, 63 cervix carcinoma, 35 corpus carcinoma and 15 ovarial cancer patients the erythrocyte transketolase, gluthathione reductase and aspartate aminotransferase activity were determined as parameters for the supply with vitamin B 1 , B 2 and B 6 before and after radiotherapy. The effects of thiamine pyrophosphate determined in cancer patients were normal but the effects of flavin adenine dinucleotide and pyridoxal-5-phosphate were significantly increased compared to the controls. These results revealed radiation-induced disorders in the B 2 metabolism and tumor-induced disorders in the B 6 metabolism. Both disorders can be avoided by treatment with vitamin B complex. (author)

Effect of wheat and Miscanthus straw biochars on soil enzymatic activity, ecotoxicity, and plant yield

Science.gov (United States)

Mierzwa-Hersztek, Monika; Gondek, Krzysztof; Klimkowicz-Pawlas, Agnieszka; Baran, Agnieszka

2017-07-01

The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

Neutron diffraction studies on GdB{sub 6} and TbB{sub 6} powders

Energy Technology Data Exchange (ETDEWEB)

Luca, S.E.; Amara, M.; Galera, R.M.; Givord, F.; Granovsky, S.; Isnard, O.; Beneu, B

2004-07-15

We report here the first powder neutron diffraction study of GdB{sub 6} and TbB{sub 6}. GdB{sub 6} and TbB{sub 6} order antiferromagnetically at 15 and 21 K, respectively. In both compounds the transition at T{sub N} is of the first order. Moreover GdB{sub 6} presents a second spontaneous magnetic transition at T{sup *}=8 K. The present study shows that, in both compounds, the magnetic propagation vectors belong to the <((1)/(4)) ((1)/(4)) ((1)/(2))> star and that the direction of the magnetic moment is perpendicular to the ((1)/(2)) component of the wave-vector in GdB{sub 6}, while in TbB{sub 6} the moment is parallel to it. The deduced low-temperature values of the magnetic moments agree with those of the respective rare-earth trivalent ions.

WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria.

KAUST Repository

Abdallah, Abdallah

2018-04-09

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria.

KAUST Repository

Abdallah, Abdallah; Weerdenburg, Eveline; Guan, Qingtian; Ummels, Roy; Borggreve, S; Adroub, Sabir; Malas, Tareq; Naeem, Raeece; Zhang, Huoming; Otto, Thomas; Bitter, Wilbert; Pain, Arnab

2018-01-01

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

Vanillin Analogues o-Vanillin and 2,4,6-Trihydroxybenzaldehyde Inhibit NFĸB Activation and Suppress Growth of A375 Human Melanoma.

Science.gov (United States)

Marton, Annamária; Kúsz, Erzsébet; Kolozsi, Csongor; Tubak, Vilmos; Zagotto, Giuseppe; Buzás, Krisztina; Quintieri, Luigi; Vizler, Csaba

2016-11-01

Constitutive activation of nuclear factor kappa-B (NFĸB) is a hallmark of various cancer types, including melanoma. Chemotherapy may further increase tumour NFĸB activity, a phenomenon that, in turn, exacerbates drug resistance. This study aimed at preliminary screening of a panel of aromatic aldehydes, including vanillin, for cytotoxicity and suppression of tumour cell NFĸB activity. The cytotoxic and NFĸB-inhibitory effects of 10 aromatic aldehydes, including vanillin, were investigated in cultured A375 human melanoma cells. Each compound was assayed alone and in combination with the model NFĸB-activating drug doxorubicin. The most promising analogues were then tested alone and in combination with 4-hydroperoxycyclophosphamide in vitro, and with cyclophosphamide in mice bearing A375 xenografts. The vanillin analogues o-vanillin and 2,4,6-trihydroxybenzaldehyde exhibited cytotoxicity against cultured A375 cells, and inhibited doxorubicin- and 4-hydroperoxycyclophosphamide-induced NFĸB activation. They also suppressed A375 cell growth in mice. o-vanillin and 2,4,6-trihydroxybenzaldehyde deserve further evaluation as potential anticancer drugs. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

International Nuclear Information System (INIS)

Tsuyama, Naohiro; Danjoh, Inaho; Otsuyama, Ken-ichiro; Obata, Masanori; Tahara, Hidetoshi; Ohta, Tsutomu; Ishikawa, Hideaki

2005-01-01

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

Science.gov (United States)

Krute, Christina N; Rice, Kelly C; Bose, Jeffrey L

2017-03-01

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in Staphylococcus aureus In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB , saeR , and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I ( coa ) and class II ( hla ) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase

Chemical Characterization, Antioxidant and Enzymatic Activity of Brines from Scandinavian Marinated Herring Products

DEFF Research Database (Denmark)

Gringer, Nina; Osman, Ali; Nielsen, Henrik Hauch

2014-01-01

Brines generated during the last marination step in the production of marinated herring (Clupea harengus) were chemically characterized and analyzed for antioxidant and enzyme activities. The end-products were vinegar cured, spice cured and traditional barrel-salted herring with either salt...... or spices. The chemical characterization encompassed pH, dry matter, ash, salt, fatty acids, protein, polypeptide pattern, iron and nitrogen. The antioxidant activity was tested with three assays measuring: iron chelation, reducing power and radical scavenging activity. The enzymatic activity for peroxidase...

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

Directory of Open Access Journals (Sweden)

Takuji Oyama

Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

Science.gov (United States)

Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

2013-01-01

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

Protein-Rich Fraction of Cnidoscolus urens (L. Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

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Yamara A. S. de Menezes

2014-03-01

Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Science.gov (United States)

Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

2016-04-01

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

Science.gov (United States)

Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

2018-01-01

Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

Science.gov (United States)

Guerra, Nelson P.; Pastrana Castro, Lorenzo

2012-01-01

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

Hepatitis B Virus Middle Protein Enhances IL-6 Production via p38 MAPK/NF-κB Pathways in an ER Stress-Dependent Manner.

Directory of Open Access Journals (Sweden)

Yang-Xia Li

Full Text Available During hepatitis B virus (HBV infection, three viral envelope proteins of HBV are overexpressed in the endoplasmic reticulum (ER. The large S protein (LHBs and truncated middle S protein (MHBst have been documented to play roles in regulating host gene expression and contribute to hepatic disease development. As a predominant protein at the ultrastructural level in biopsy samples taken from viremic patients, the role of the middle S protein (MHBs remains to be understood despite its high immunogenicity. When we transfected hepatocytes with an enhanced green fluorescent protein (EGFP-tagged MHBs expressing plasmid, the results showed that expression of MHBs cause an upregulation of IL-6 at the message RNA and protein levels through activating the p38 mitogen-activated protein kinase (p38 MAPK and nuclear factor-kappa B (NF-κB pathways. The use of specific inhibitors of the signaling pathways can diminish this upregulation. The use of BAPTA-AM attenuated the stimulation caused by MHBs. We further found that MHBs accumulated in the endoplasmic reticulum and increased the amount of glucose regulated protein 78 (GRP78/BiP. Our results provide a possibility that MHBs could be involved in liver disease progression.

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

Science.gov (United States)

Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

2016-03-01

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. Copyright © 2016 Elsevier Inc. All rights reserved.

Cre-dependent DNA recombination activates a STING-dependent innate immune response

Science.gov (United States)

Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit

2016-01-01

Abstract Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

Science.gov (United States)

Li, Xian; Lee, Youn Ju; Jin, Fansi; Park, Young Na; Deng, Yifeng; Kang, Youra; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Chang, Young-Chae; Ko, Hyun-Jeong; Kim, Cheorl-Ho; Murakami, Makoto; Chang, Hyeun Wook

2017-07-25

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

International Nuclear Information System (INIS)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue; Tsuchihashi, Kenji; Ueda, Shiho; Masuko, Takashi; Saya, Hideyuki; Nagano, Osamu

2016-01-01

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

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Schmidt, A; Schreiner, M G; Mayer, H K

2017-06-02

As the formation of pyridoxal phosphate, the active cofactor of vitamin B 6 , is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B 6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B 2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B 6 and B 2 vitamers. In cow's milk, vitamin B 6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B 6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69μmolL -1 . Vitamin B 2 was present as riboflavin and its phosphate ester up to 12.86μmolL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous high performance liquid chromatographic analysis of vitamins B1, B2 and B6 in royal jelly

Directory of Open Access Journals (Sweden)

Presoto Ana Elisa F

2004-01-01

Full Text Available Royal jelly is used as a food supplement, popularly known as rich in B vitamins. The present work has two objectives: firstly, to apply simultaneous quantitative determination by High Performance Liquid Chromatography of thiamin (vitamin B1, riboflavin (vitamin B2 and pyridoxine (vitamin B6 and secondly to compare the obtained data with the Dietary Reference Intake (DRI values. The values obtained showed no thiamin, a range from 20 to 171 ng g-1 of riboflavin and from 408 to 2 188 ng g-1 of pyridoxine in royal jelly. According to the Food and Nutrition Board (2000, the DRI of these vitamins varies from 0.2-1.4 mg for thiamin; 0.3-1.6 mg for riboflavin and 0.1-2.0 mg for pyridoxine, depending on age and sex. According to these recommendations, royal jelly is not a good source of vitamins B1, B2 or B6 as these vitamins appear only on order of ng g-1. The proposed method can be used in routine analysis for royal jelly, having the advantage of being simple, fast and reliable.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

Science.gov (United States)

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Purification and enzymatic characterization of a novel β-1,6-glucosidase from Aspergillus oryzae.

Science.gov (United States)

Watanabe, Akira; Suzuki, Moe; Ujiie, Seiryu; Gomi, Katsuya

2016-03-01

In this study, among the 10 genes that encode putative β-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding β-1,6-glucosidase with a substrate specificity for gentiobiose. The transformant harboring AO090038000425, which we named bglH, was overexpressed under the control of the improved glaA gene promoter to form a small clear zone around the colony in a plate assay using 4-methylumbelliferyl β-d-glucopyranoside as the fluorogenic substrate for β-glucosidase. We purified BglH to homogeneity and enzymatically characterize this enzyme. The thermal and pH stabilities of BglH were higher than those of other previously studied A. oryzae β-glucosidases, and BglH was stable over a wide temperature range (4°C-60°C). BglH was inhibited by Hg(2+), Zn(2+), glucono-δ-lactone, glucose, dimethyl sulfoxide, and ethanol, but not by ethylenediaminetetraacetic acid. Interestingly, BglH preferentially hydrolyzed gentiobiose rather than other oligosaccharides and aryl β-glucosides, thereby demonstrating that this enzyme is a β-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of β-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable β-glucosidase such as BglH for reducing the bitter taste of gentiobiose, and thus for controlling the sweetness of starch hydrolysates in the food industry via genome mining. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Postprandial incretin and islet hormone responses and dipeptidyl-peptidase 4 enzymatic activity in patients with maturity onset diabetes of the young

DEFF Research Database (Denmark)

Østoft, Signe Harring; Bagger, Jonatan Ising; Hansen, Torben

2015-01-01

Objective: The role of the incretin hormones in the pathophysiology of maturity onset diabetes of the young (MODY) is unclear. Design: We studied the postprandial plasma responses of glucagon, incretin hormones (glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP......)), and dipeptidyl-peptidase 4 (DPP-4) enzymatic activity in patients with glucokinase (GCK)-diabetes (MODY2), hepatocyte nuclear factor 1α (HNF1A)-diabetes (MODY3), and in matched healthy individuals (CTRLs). Subjects and methods: Ten patients with GCK-diabetes (age: 43±5 years; BMI: 24±2 kg/m2; FPG: 7.1±0.3 mmol....../l: HbA1c: 6.6±0.2%), 10 patients with HNF1A-diabetes (age: 31±3 years (mean ± SEM); body mass index (BMI): 24±1 kg/m2; fasting plasma glucose (FPG): 8.9±0.8 mmol/l; haemoglobin A1c (HbA1c): 7.0±0.3%), and 10 CTRLs (age: 40±5 years; BMI: 24±1 kg/m2; FPG: 5.1±0.1 mmol/l; HbA1c: 5.3±0.1%) were examined...

Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway.

Science.gov (United States)

Mao, Xiao-Yuan; Zhou, Hong-Hao; Li, Xi; Liu, Zhao-Qian

2016-08-01

Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway.

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

Science.gov (United States)

Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

2015-07-30

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Transactivation mediated by B-Myb is dependent on TAF(II)250.

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Bartusel, Thorsten; Klempnauer, Karl-Heinz

2003-05-15

B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. We have employed the hamster cell line ts13 as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells.

Enzymatic determination of rare earth elements using pyrophosphatases

International Nuclear Information System (INIS)

Shekhovtsova, T.N.; Pirogova, S.V.; Fedorova, O.M.; Dolmanova, I.F.; Bajkov, A.A.

1993-01-01

A highly sensitive(determination limit 8x10 -6 -4x10 -4 μ g/m) and selective enzymatic method for determination of rare earth elements has been developed. The method is based on inhibition action of rare earths on the catalytic activity of pyrophosphates isolated from bakery geast and E.Coli. The mechanism of the rare earth element action, corresponding to competitive inhibition, has been established

Multicenter evaluation of an enzymatic method for glycated albumin.

Science.gov (United States)

Paleari, Renata; Bonetti, Graziella; Callà, Cinzia; Carta, Mariarosa; Ceriotti, Ferruccio; Di Gaetano, Nicola; Ferri, Marilisa; Guerra, Elena; Lavalle, Gabriella; Cascio, Claudia Lo; Martino, Francesca Gabriela; Montagnana, Martina; Moretti, Marco; Santini, Gabriele; Scribano, Donata; Testa, Roberto; Vero, Anna; Mosca, Andrea

2017-06-01

The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA 1c test is not reliable. We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

Quercetin inhibits the invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

Science.gov (United States)

Zhang, Xian-Ming; Huang, Shao-Peng; Xu, Qiang

2004-01-01

On the basis of the inhibitory effect of quercetin on the invasion of melanoma B16-BL6 cells previously reported by us, the mechanisms of quercetin-mediated inhibition of invasion were further investigated in the present study. The ability of B16-BL6 cells to invade and migrate was evaluated in terms of the numbers of cells penetrating a reconstituted basement membrane in the Transwell coculture system. The relative levels and activities of matrix metalloproteinase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and quantified using LabWorks 4.0 software. The quercetin-mediated inhibition of invasion was partially blocked by phorbol-12,13-dibutyrate (PDB), a PKC (protein kinase C) activator, and by doxorubicin, a PKC inhibitor. Only the proforms of MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected by gelatin zymography. Quercetin dose-dependently decreased the gelatinolytic activity of pro-MMP-9. Doxorubicin also markedly reversed the quercetin-induced decrease. Quercetin showed a dose-dependent antagonism of increases in gelatinolytic activity of pro-MMP-9 induced by PDB and free fatty acid (another PKC activator). Together with the report that quercetin directly reduces PKC activity, the results reported here suggest that quercetin may inhibit the invasion of B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

Science.gov (United States)

Zhang, X; Xu, Q; Saiki, I

2000-01-01

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

Science.gov (United States)

Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

2017-06-21

In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B ( 1 ) contains an additional phenolic hydroxy function at C-6 and exhibits an IC 50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin ( 2 ) did not show any inhibition of this enzymatic activity. Asperentin B ( 1 ) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.

Automatic single- and multi-label enzymatic function prediction by machine learning

Directory of Open Access Journals (Sweden)

Shervine Amidi

2017-03-01

Full Text Available The number of protein structures in the PDB database has been increasing more than 15-fold since 1999. The creation of computational models predicting enzymatic function is of major importance since such models provide the means to better understand the behavior of newly discovered enzymes when catalyzing chemical reactions. Until now, single-label classification has been widely performed for predicting enzymatic function limiting the application to enzymes performing unique reactions and introducing errors when multi-functional enzymes are examined. Indeed, some enzymes may be performing different reactions and can hence be directly associated with multiple enzymatic functions. In the present work, we propose a multi-label enzymatic function classification scheme that combines structural and amino acid sequence information. We investigate two fusion approaches (in the feature level and decision level and assess the methodology for general enzymatic function prediction indicated by the first digit of the enzyme commission (EC code (six main classes on 40,034 enzymes from the PDB database. The proposed single-label and multi-label models predict correctly the actual functional activities in 97.8% and 95.5% (based on Hamming-loss of the cases, respectively. Also the multi-label model predicts all possible enzymatic reactions in 85.4% of the multi-labeled enzymes when the number of reactions is unknown. Code and datasets are available at https://figshare.com/s/a63e0bafa9b71fc7cbd7.

NFκB-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1.

Science.gov (United States)

Nordén, Rickard; Samuelsson, Ebba; Nyström, Kristina

2017-11-01

Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus specific mechanism for transcriptional activation. The aim of this study was to define the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT-gene cluster on chromosome 19 in fibroblasts was specific, not involving adjacent genes. Moreover, inhibition of NFκB signaling through panepoxydone treatment significantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65, in herpes simplex virus type 1 infected fibroblasts. NFκB and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentified, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NFκB was not sufficient to induce the fucosyltransferase encoding gene cluster in uninfected cells. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cerebral ischemia is exacerbated by extracellular nicotinamide phosphoribosyltransferase via a non-enzymatic mechanism.

Directory of Open Access Journals (Sweden)

Bing Zhao

Full Text Available Intracellular nicotinamide phosphoribosyltransferase (iNAMPT in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

Lactoferricin B-derived peptides with inhibitory effects on ECE-dependent vasoconstriction.

Science.gov (United States)

Fernández-Musoles, Ricardo; López-Díez, José Javier; Torregrosa, Germán; Vallés, Salvador; Alborch, Enrique; Manzanares, Paloma; Salom, Juan B

2010-10-01

Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an ex vivo functional assay was developed using isolated rabbit carotid artery segments. With FPS V, only four LfcinB-derived peptides induced inhibition of ECE activity, whereas the eight peptides showed ECE inhibitory effects with big ET-1 as substrate. Regarding the ex vivo assays, six LfcinB-derived peptides showed inhibition of big ET-1-induced, ECE-dependent vasoconstriction. A positive correlation between the inhibitory effects of LfcinB-derived peptides on ECE activity when using big ET-1 and the inhibitory effects on ECE-dependent vasoconstriction was shown. ECE-independent vasoconstriction induced by ET-1 was not affected, thus discarding effects of LfcinB-derived peptides on ET(A) receptors or intracellular signal transduction mechanisms. In conclusion, a combined in vitro and ex vivo method to assess the effects of potentially antihypertensive peptides on the ET system has been developed and applied to show the inhibitory effects on ECE-dependent vasoconstriction of six LfcinB-derived peptides, five of which were dual vasopeptidase (ACE/ECE) inhibitors. Copyright © 2010 Elsevier Inc. All rights reserved.

Design and discovery of thioether and nicotinamide containing sorafenib analogues as multikinase inhibitors targeting B-Raf, B-RafV600E and VEGFR-2.

Science.gov (United States)

Sun, Shaofeng; He, Zuopeng; Huang, Mindong; Wang, Ningning; He, Zongzhong; Kong, Xiangkai; Yao, Jianwen

2018-04-03

New sorafenib derivatives containing thioether and nicotinamide moiety were designed and synthesized as B-Raf, B-Raf V600E and VEGFR-2 multikinase inhibitors. Their in vitro enzymatic inhibitory activities against B-Raf, B-Raf V600E and VEGFR-2 and their antiproliferative activities against HCT-116 and B16BL6 cell lines were evaluated and described. Most of the compounds showed potent activities against both cell lines and specific kinases. Compounds a1, b1 and c4, which exhibited the most potent inhibitory activities against B-Raf with IC 50 of 21 nM, 27 nM and 17 nM, B-Raf V600E with IC 50 of 29 nM, 28 nM and 16 nM, VEGFR-2 with IC 50 of 84 nM, 46 nM and 63 nM, respectively, and good antiproliferative activities, also demonstrated competitive antiangiogenic activities to sorafenib in in vitro HUVEC tube formation assay. Copyright © 2018 Elsevier Ltd. All rights reserved.

Site-different structures from dilithium hexaboride (Li2b6) to dimagnesium hexaboride (Mg2B6) by first-principles

International Nuclear Information System (INIS)

Aydın, Sezgin

2013-01-01

Highlights: •All structures are thermodynamically stable. All structures are metallic. •Boron sub-lattice have negative-charged atoms and more covalent bonds. •The inter-octahedral binding is more covalent than inner-octahedral binding. •All structures are also mechanically stable. -- Abstract: The structural, mechanical, electronic and bonding properties of dilithium hexaboride (Li 2 B 6 ) and isostructural hypothetic compounds obtained by replacing Li atoms in different sites to magnesium atoms have been investigated by first-principles density functional pseudopotential plane–wave calculations. It is shown that calculated lattice parameters of Li 2 B 6 agree with the experimental results. All of designed hypothetical structures have negative formation enthalpies, thus all of them are thermodynamically stable and the most stable structure is Mg 2 B 6 . At the same time, from calculated single crystal elastic constants, it is shown that all structures are mechanically stable and related mechanical properties such as bulk, shear and Young moduli are calculated. It is shown that adding magnesium to the structure of Li 2 B 6 is decreasing values of the moduli. Further, hardnesses of the structures are determined theoretically and it is obtained that hardness exhibits same trend with the moduli. From electronic structure calculations including band structure and site-dependent density of states, all structures are metallic, and fully magnesium substituted structure (Mg 2 B 6 ) has the highest metallicity among the structures. Additionally, bonding nature of the structures are analyzed by using electron density maps, Mulliken atomic charges and bond overlap populations

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

Science.gov (United States)

Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun, E-mail: lj@ahmu.edu.cn

2016-02-01

Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

International Nuclear Information System (INIS)

Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun

2016-01-01

Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.

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Carmen H Coxon

Full Text Available Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM and an immunoreceptor tyrosine-based switch motif (ITSM. The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50 for both CRP and collagen.

Efficient Enzymatic Routes for the Synthesis of New Eight-membered Cyclic β-Amino Acid and β-Lactam Enantiomers

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Enikő Forró

2017-12-01

Full Text Available Efficient enzymatic resolutions are reported for the preparation of new eight-membered ring-fused enantiomeric β-amino acids [(1R,2S-9 and (1S,2R-9] and β-lactams [(1S,8R-3, (1R,8S-3 (1S,8R-4 and (1R,8S-7], through asymmetric acylation of (±-4 (E > 100 or enantioselective hydrolysis (E > 200 of the corresponding inactivated (±-3 or activated (±-4 β-lactams, catalyzed by PSIM or CAL-B in an organic solvent. CAL-B-catalyzed ring cleavage of (±-6 (E > 200 resulted in the unreacted (1S,8R-6, potential intermediate for the synthesis of enantiomeric anatoxin-a. The best strategies, in view of E, reaction rate and product yields, which underline the importance of substrate engineering, are highlighted.

Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

Science.gov (United States)

Kunze, Cindy; Bommer, Martin; Hagen, Wilfred R; Uksa, Marie; Dobbek, Holger; Schubert, Torsten; Diekert, Gabriele

2017-07-03

The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B 12 -dependent biochemistry and represents an effective mode of RDase catalysis.

Cocoa consumption reduces NF-κB activation in peripheral blood mononuclear cells in humans.

Science.gov (United States)

Vázquez-Agell, M; Urpi-Sarda, M; Sacanella, E; Camino-López, S; Chiva-Blanch, G; Llorente-Cortés, V; Tobias, E; Roura, E; Andres-Lacueva, C; Lamuela-Raventós, R M; Badimon, L; Estruch, R

2013-03-01

Epidemiological studies have demonstrated an association between high-polyphenol intake and reduced incidence of atherosclerosis. The healthy effects of cocoa-polyphenols may be due to their antioxidant and anti-inflammatory actions, although the exact mechanisms are unknown and depend on the matrix in which cocoa-polyphenols are delivered. Nuclear factor κB (NF-κB) is a key molecule in the pathophysiology of atherosclerosis involved in the regulation of adhesion molecules(AM) and cytokine expression and its activation is the first step in triggering the inflammatory process. The aim of this study was to evaluate the effect of acute cocoa consumption in different matrices related to the bioavailability of cocoa-polyphenols in NF-κB activation and the expression of AM. Eighteen healthy volunteers randomly received 3 interventions: 40g of cocoa powder with milk (CM), with water (CW), and only milk (M). NF-κB activation in leukocytes and AM (sICAM, sVCAM, E-selectin) were measured before and 6h after each intervention. Consumption of CW significantly decreased NF-κB activation compared to baseline and to CM (P cocoa intake may depend on the bioavailability of bioactive compounds and may be mediated at least in part by the modulation of NF-κB activation and downstream molecules reinforcing the link between cocoa intake and health. Copyright © 2011 Elsevier B.V. All rights reserved.

Role of Erbin in ErbB2-dependent breast tumor growth

Science.gov (United States)

Tao, Yanmei; Shen, Chengyong; Luo, Shiwen; Traoré, Wilfried; Marchetto, Sylvie; Santoni, Marie-Josée; Xu, Linlin; Wu, Biao; Shi, Chao; Mei, Jinghong; Bates, Ryan; Liu, Xihui; Zhao, Kai; Xiong, Wen-Cheng; Borg, Jean-Paul; Mei, Lin

2014-01-01

ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), a receptor tyrosine kinase of the ErbB family, is overexpressed in around 25% of breast cancers. In addition to forming a heterodimer with other ErbB receptors in response to ligand stimulation, ErbB2 can be activated in a ligand-independent manner. We report here that Erbin, an ErbB2-interacting protein that was thought to act as an antitumor factor, is specifically expressed in mammary luminal epithelial cells and facilitates ErbB2-dependent proliferation of breast cancer cells and tumorigenesis in MMTV-neu transgenic mice. Disruption of their interaction decreases ErbB2-dependent proliferation, and deletion of the PDZ domain in Erbin hinders ErbB2-dependent tumor development in MMTV-neu mice. Mechanistically, Erbin forms a complex with ErbB2, promotes its interaction with the chaperon protein HSP90, and thus prevents its degradation. Finally, ErbB2 and Erbin expression correlates in human breast tumor tissues. Together, these observations establish Erbin as an ErbB2 regulator for breast tumor formation and progression. PMID:25288731

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

KAUST Repository

Ayo, Begoñ a; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñ igo; Bañ a, Zuriñ e; Unanue, Marian; Gasol, Josep M.; Duarte, Carlos M.; Iriberri, Juan

2017-01-01

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature-dependence has not yet been evaluated. Here we report a global assessment of the temperature-sensitivity, as represented by the activation energies (Ea ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen and phosphorus, respectively. These Ea were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting Ea in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. This article is protected by copyright. All rights reserved.

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

KAUST Repository

Ayo, Begoña

2017-06-08

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature-dependence has not yet been evaluated. Here we report a global assessment of the temperature-sensitivity, as represented by the activation energies (Ea ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen and phosphorus, respectively. These Ea were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting Ea in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. This article is protected by copyright. All rights reserved.

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities.

Science.gov (United States)

Ayo, Begoña; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñigo; Baña, Zuriñe; Unanue, Marian; Gasol, Josep M; Duarte, Carlos M; Iriberri, Juan

2017-10-01

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO 2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature-sensitivity, as represented by the activation energies (E a ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E a were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting E a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. © 2017 John Wiley & Sons Ltd.

Age-dependent Hepatic UDP-glucuronosyltransferase Gene Expression and Activity in Children

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Elizabeth Neumann

2016-11-01

Full Text Available ABSTRACTUDP-glucuronosyltransferases (UGTs are important phase II drug metabolism enzymes. The aim of this study was to explore the relationship between age and changes in mRNA expression and activity of major human hepatic UGTs, as well as to understand the potential regulatory mechanism underlying this relationship. Using previously generated data, we investigated age-dependent mRNA expression levels of 11 hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17 and 16 transcription factors (AHR, AR, CAR, ESR2, FXR, GCCR, HNF1a, HNF3a, HNF3b, HNF4a, PPARA, PPARG, PPARGC, PXR, SP1, and STAT3 in liver tissue of donors (n = 38 ranging from 0 to 25 years of age. We also examined the correlation between age and microsomal activities using 14 known UGT drug substrates in the liver samples (n = 19 of children donors. We found a statistically significant increase (nominal p < 0.05 in the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT2B7 and UGT2B17, as well as glucuronidation activities of serotonin, testosterone, and vorinostat during the first 25 years of life. Expression of estrogen receptor 1 (ESR1 and pregnane X receptor (PXR, two strong UGT transcriptional regulators, were significantly correlated with both age and UGT mRNA expression (p ≤ 0.05. These results suggest that both UGT expression and activity increase during childhood and adolescence, possibly driven in part by hormonal signaling. Our findings may help explain inter-patient variability in response to medications among children.

[Evaluations of newborn screening program performance and enzymatic diagnosis of glucose-6-phosphate dehydrogenase deficiency in Guangzhou].

Science.gov (United States)

Tang, F; Huang, Y L; Jiang, X; Jia, X F; Li, B; Feng, Y; Chen, Q Y; Tang, C F

2018-05-02

Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PDblood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PDT, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PDblood cells were 95.5%, 97.2%, respectively. Conclusions: The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will

Microstructural characterization of LaB6-ZrB2 eutectic composites

International Nuclear Information System (INIS)

Wang Shengchang; Wei, W.J.; Zhang Litong

2003-01-01

Detail microstructure of LaB 6 -ZrB 2 composites has been characterized by TEM and HRTEM. The directionally solidified ZrB 2 fibers in LaB 6 matrix near LaB 6 -ZrB 2 eutectics present at least three growing relationship systems. In addition to previous report of [001]LaB 6 / [0001]ZrB 2 relationship, [0 anti 11]LaB 6 / [0001]ZrB 2 and [1 anti 20]LaB 6 / [0001]ZrB 2 . were identified. Different with [001]LaB 6 / [0001]ZrB 2 system, the interfaces of [0 anti 11]LaB 6 / [0001]ZrB 2 and [1 anti 20]LaB 6 / [0001]ZrB 2 . show non-coherent and clean interfaces. There is neither glassy phase nor reaction products found at the interfaces (orig.)

Astaxanthin inhibits cytokines production and inflammatory gene expression by suppressing IκB kinase-dependent nuclear factor κB activation in pre and postpartum Murrah buffaloes during different seasons

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Lakshmi Priyadarshini

2018-06-01

Full Text Available Aim: We examined regulatory function of astaxanthin on mRNA expression of nuclear factor κB (NF-κB p65, interleukin-6 (IL-6, tumor necrosis factor alpha (TNF-α, and interferon gamma (IFN-γ in peripheral blood mononuclear cells in pre and postpartum Murrah buffaloes during summer (temperature-humidity index [THI]=86; relative humidity [RH]=24 and winter (THI=58.74; RH=73 seasons. Materials and Methods: A total of 32 Murrah buffaloes apparently healthy and in their one to four parity were selected from National Dairy Research Institute herd and equally distributed randomly into four groups (control and supplemented groups of buffaloes during summer and winter season, respectively. All groups were fed according to the nutrient requirement of buffaloes (ICAR, 2013. The treatment group was supplemented with astaxanthin at 0.25 mg/kg body weight/animal/day during the period 30 days before expected date of calving and up to 30 days postpartum. Results: There was downregulation of NF-κB p65 gene in all the groups. NF-κB p65 mRNA expression was lower (p<0.05 in treatment than control group from prepartum to postpartum during summer, while mRNA expression was low only on day 21 after calving during winter season. The mRNA expression of IL-6, TNF-α, and IFN-γ was lower (p<0.05 in treatment than a control group of buffaloes during summer and winter seasons. The mRNA expression of NF-κB p65, IL-6, TNF-α, and IFN-γ was higher (p<0.05 in summer than in winter seasons. Conclusion: The xanthophyll carotenoid astaxanthin a reddish-colored C-40 compound is a powerful broad-ranging antioxidant that naturally occurs in a wide variety of living organisms, such as microalgae, fungi, crustaceans, and complex plants. Astaxanthin blocked nuclear translocation of NF-κB p65 subunit and IκBa degradation, which correlated with its inhibitory effect on IκB kinase (IKK activity. These results suggest that astaxanthin, probably due to its antioxidant activity

Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

Science.gov (United States)

Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

2015-06-01

An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

STUDY ON QUALITY PARAMETERS AND ENZYMATIC ACTIVITY OF GRAIN MILL PRODUCTS REGION IN TRANSYLVANIA

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Glevitzky Mirel

2011-07-01

Full Text Available This paper aims at determining the main quality parameters of grain mill products in the Transylvania region, also studying and emphasizing the enzymatic activity of flour. Determination of quality characteristics of grain mill products entails establishing physical, chemical and sensory parameters and assessing them against the limits imposed by law. Analysis was performed on samples formed by mixing basic medium extracted from different batches. Incremental size, sampling tools, how to extract them, the training sample and laboratory environments, packaging and labeling of samples were performed according to STAS 1068 69. Determination of the fall (Falling Number, an empirical test that relies on the ability of endogenous ?-amylase to reduce viscosity of the treated warm flour suspension is used, large scale milling and bakery industry to predict and assess the Baking quality of flour. In sprouted wheat, characterised by a low Falling number, dextrin produced by the action of ?-amylase leads to a sticky bread core. Experiments suggest that the values fall turnover (FN does not shrink in direct proportion to the percentage of germinating seeds. Amylolytic activity depends on the stage of sprouting of grains. Lack of ?-amylase activity can be corrected by adding malt grain ?-amylase or fungal ?-amylase.

Eggshell membrane hydrolyzates activate NF-κB in vitro: possible implications for in vivo efficacy

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Ruff KJ

2015-02-01

Full Text Available Kevin J Ruff,1 Paul L Durham,2 Austin O’Reilly,2 F Daniel Long1 1ESM Technologies, LLC, Carthage, MO, USA; 2Center for Biomedical and Life Sciences, Missouri State University, Springfield, MO, USA Purpose: Eggshell membrane (ESM has been shown to contain naturally occurring bioactive components, and biological activities such as reducing proinflammatory cytokines, liver fibrosis, and joint pain in osteoarthritis sufferers have also been reported for ESM matrix as a whole. Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB is a signaling protein found in the cytoplasm of nearly all human and animal cell types and is a primary regulator of immune function. The studies reported herein were designed to investigate the possible role that NF-κB activity might play in the reported biological activities of ESM. Methods: Three ESM hydrolyzates produced via fermentation, enzymatic, or chemical hydrolysis were evaluated in vitro in either human peripheral blood mononuclear cell or THP-1 (human leukemic monocyte cell cultures for NF-κB activity following 4-hour exposure. The hydrolyzates were compared with untreated control cells or cells incubated with lipopolysaccharide or ascorbic acid. The source of ESM activity was also evaluated. Results: NF-κB levels were increased above levels found in untreated cells at all three dilutions (1:100, 1:1,000, and 1:10,000 for the fermentation hydrolyzate of ESM (ESM-FH (P=0.021, P=0.020, P=0.009, respectively in peripheral blood mononuclear cells. The enzymatic hydrolyzate of ESM (ESM-EH also produced statistically significant levels of activated NF-κB at the 1:100 and 1:1,000 dilutions (P=0.004, P=0.006, respectively but fell just shy of significance at the 1:10,000 dilution (P=0.073. Similarly, ESM-FH (P=0.021, P=0.002 and ESM-EH (P=0.007, P=0.007 activated NF-κB in THP-1 cells at 1:1,000 and 1:10,000 dilutions, respectively. The chemical hydrolyzate of ESM (ESM-CH showed statistically

Impact of Heavy Metals in Enzymatic Activity of Soils from Hidalgo, Mexico

International Nuclear Information System (INIS)

Reyes-Ortigoza, A. L.; Reyes-Solis, I. E.; Galicia-Palacios, M. S.; Montiel-Arteaga, S.

2009-01-01

The soils from Valle of Mezquital, Hidalgo, Mexico have been irrigated with waste waters from Mexico City for more than 88 years. the present investigation was made in order to know the relationship between heavy metal contents and time of irrigation with waste waters and production of CO 2 and enzymatic activity in soils from Valle Mezquital for knowing the disponibility of nutrients and degradation of soils. (Author)

Reversible pH-dependent activation/inactivation of CF(1-ATPase of spinach chloroplasts

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A. P. Khomochkin

2017-08-01

Full Text Available The aim of the work was to study the reverse pH-dependent regulation of the enzymatic activity of the catalytic part of ATP synthase (EC 3.6.3.14 of chloroplast – coupling factor CF1. It was shown that the short-term incubation of isolated CF1 in the media with pH 4.5 or 3.5 leads to inactivation of Ca2+-ATPase, which is rapidly (t1/2 ~ 1 min restored in the medium containing 0.5-10 mM bicarbonate at pH 7.8. After acid treatment, the rate of Mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С. The increase in Ca2+– and Mg2+-АТР activity of CF1 associated with the addition of NaHCO3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. The obtained results suggest the existence of the bound bicarbonate in the CF1 structure, which apparently participates in proton transfer.

Chondroitin sulphate extracted from antler cartilage using high hydrostatic pressure and enzymatic hydrolysis

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Chong-Tai Kim

2014-12-01

Full Text Available Chondroitin sulphate (CS, a major glycosaminoglycan, is an essential component of the extracellular matrix in cartilaginous tissues. Wapiti velvet antlers are a rich source of these molecules. The purpose of the present study was to develop an effective isolation procedure of CS from fresh velvet antlers using a combination of high hydrostatic pressure (100 MPa and enzymatic hydrolysis (papain. High CS extractability (95.1 ± 2.5% of total uronic acid was obtained following incubation (4 h at 50 °C with papain at pH 6.0 in 100 MPa compared to low extractability (19 ± 1.1% in ambient pressure (0.1 MPa. Antler CS fractions were isolated by Sephacryl S-300 chromatography and identified by western blot using an anti-CS monoclonal antibody. The antler CS fraction did not aggregate with hyaluronic acid in CL-2B chromatography and possessed DPPH radical scavenging activity at 78.3 ± 1.5%. The results indicated that high hydrostatic pressure and enzymatic hydrolysis procedure may be a useful tool for the isolation of CS from antler cartilaginous tissues.

bFGF influences human articular chondrocyte differentiation

DEFF Research Database (Denmark)

Schmal, H; Zwingmann, J; Fehrenbach, M

2007-01-01

BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro......FGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained...... by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential...

A singular enzymatic megacomplex from Bacillus subtilis.

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Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

2007-01-02

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

CYP2D6 genotype dependent oxycodone metabolism in postoperative patients.

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Stamer, Ulrike M; Zhang, Lan; Book, Malte; Lehmann, Lutz E; Stuber, Frank; Musshoff, Frank

2013-01-01

The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone's metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption. Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA) for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele), HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity), EM (extensive metabolizers, normal CYP2D6 activity) and UM (ultrarapid metabolizers, increased CYP2D6 activity). Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide. Metabolism differed between CYP2D6 genotypes. Mean (95%-CI) oxymophone/oxycodone ratios were 0.10 (0.02/0.19), 0.13 (0.11/0.16), 0.18 (0.16/0.20) and 0.28 (0.07/0.49) in PM, HZ/IM, EM and UM, respectively (p = 0.005). Oxycodone consumption up to the 12(th) hour was highest in PM (p = 0.005), resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8); EM and UM 2.2 (2.1/2.3); p<0.001). Pain scores did not differ between genotypes. In this postoperative setting, the number of functionally active CYP2D6 alleles had an impact

CYP2D6 genotype dependent oxycodone metabolism in postoperative patients.

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Ulrike M Stamer

Full Text Available BACKGROUND: The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone's metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption. METHODS: Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele, HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity, EM (extensive metabolizers, normal CYP2D6 activity and UM (ultrarapid metabolizers, increased CYP2D6 activity. Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide. RESULTS: Metabolism differed between CYP2D6 genotypes. Mean (95%-CI oxymophone/oxycodone ratios were 0.10 (0.02/0.19, 0.13 (0.11/0.16, 0.18 (0.16/0.20 and 0.28 (0.07/0.49 in PM, HZ/IM, EM and UM, respectively (p = 0.005. Oxycodone consumption up to the 12(th hour was highest in PM (p = 0.005, resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8; EM and UM 2.2 (2.1/2.3; p<0.001. Pain scores did not differ between genotypes. CONCLUSIONS: In this postoperative setting, the number of

Novel differential refractometry study of the enzymatic degradation kinetics of poly(ethylene oxide)-b-poly(epsilon-caprolactone) particles dispersed in water.

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Lam, HiuFung; Gong, Xiangjun; Wu, Chi

2007-02-22

A poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) diblock copolymer was micronized into small micelle-like particles (approximately 80 nm) via dialysis-induced microphase inversion. The enzymatic biodegradation of the PCL portion of these particles in water was in situ investigated inside a recently developed novel differential refractometer. Using this refractometry method, we were able to monitor the real-time biodegradation via the refractive index change (Deltan) of the dispersion because Deltan is directly proportional to the particle mass concentration. We found that the degradation rate is proportional to either the polymer or enzyme concentration. Our results directly support previous speculation on the basis of the light-scattering data that the biodegradation follows the first-order kinetics for a given enzyme concentration. This study not only leads to a better understanding of the enzymatic biodegradation of PCL, but also demonstrates a novel, rapid, noninvasive, and convenient way to test the degradability of polymers.

A novel CYP27B1 mutation causes a feline vitamin D-dependent rickets type IA.

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Grahn, Robert A; Ellis, Melanie R; Grahn, Jennifer C; Lyons, Leslie A

2012-08-01

A 12-week-old domestic cat presented at a local veterinary clinic with hypocalcemia and skeletal abnormalities suggestive of rickets. Osteomalacia (rickets) is a disease caused by impaired bone mineralization leading to an increased prevalence of fractures and deformity. Described in a variety of species, rickets is most commonly caused by vitamin D or calcium deficiencies owing to both environmental and or genetic abnormalities. Vitamin D-dependent rickets type 1A (VDDR-1A) is a result of the enzymatic pathway defect caused by mutations in the 25-hydroxyvitamin D(3)-1-alpha-hydroxylase gene [cytochrome P27 B1 (CYP27B1)]. Calcitriol, the active form of vitamin D(3), regulates calcium homeostasis, which requires sufficient dietary calcium availability and correct hormonal function for proper bone growth and maintenance. Patient calcitriol concentrations were low while calcidiol levels were normal suggestive of VDDR-1A. The entire DNA coding sequencing of CYP27B1 was evaluated. The affected cat was wild type for previously identified VDDR-1A causative mutations. However, six novel mutations were identified, one of which was a nonsense mutation at G637T in exon 4. The exon 4 G637T nonsense mutation results in a premature protein truncation, changing a glutamic acid to a stop codon, E213X, likely causing the clinical presentation of rickets. The previously documented genetic mutation resulting in feline VDDR-1A rickets, as well as the case presented in this research, result from novel exon 4 CYP27B1 mutations, thus exon 4 should be the initial focus of future sequencing efforts.

Influence of season, environment and feeding habits on the enzymatic activity of peptidase and β-glucosidase in the gastrointestinal tract of two Siluriformes fishes (Teleostei

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Silvana Duarte

2013-06-01

Full Text Available The enzymatic activities involved in the digestion of proteins and carbohydrates were compared among three organs of the digestive track of two Siluriformes fish species, and between two areas: a reservoir, and an area downriver of it. Our aim was to test the hypothesis that the digestive organs of species with varied feeding habits have different enzymatic activities, and that the enzymatic activity differs among seasons and environmental conditions. The iliophagous/herbivorous species Hypostomus auroguttatus Kner, 1854 had higher trypsin-like, chymotrypsin-like peptidase and β-glucosidase activity in the intestine when compared with the omnivorous species Pimelodus maculatus Lacepède, 1803, whereas the latter had more hepatic trypsin-like activity than the former. The peak of activity of the three enzymes in H. auroguttatus was recorded in the winter and spring. On the other hand, P. maculatus tended to have the more prominent peptidase and β-glucosidase activity in the summer, and the smallest in the winter. The intestine of H. auroguttatus had higher enzymatic (trypsin, chymotrypsin and β-glucosidase activity than the stomach and the liver. For P. maculatus, the highest β-glucosidase activity was found in the liver. The enzymatic activity of H. aurogutattus did not differ between lotic and lentic systems, whereas P. maculatus had comparatively higher stomach and hepatic trypsin levels and hepatic chymotrypsin-like activities in the reservoir than down in the river. These findings indicate that, in H. auroguttatus, most digestive activity occurs in the intestine, which is long and adapted for the digestion of bottom-river vegetable matter and detritus. The seasons and the type of the system (lentic vs. lotic seem to affect the enzymatic activity for these two species differently, a likely consequence of their different lifestyles.

The effect of amino-acid substitutions I112P, D147E and K152N in CYP11B2 on the catalytic activities of the enzyme.

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Bechtel, Stephanie; Belkina, Natalya; Bernhardt, Rita

2002-02-01

By replacing specific amino acids at positions 112, 147 and 152 of the human aldosterone synthase (CYP11B2) with the corresponding residues from human, mouse or rat 11beta-hydroxylase (CYP11B1), we have been able to investigate whether these residues belong to structural determinants of individual enzymatic activities. When incubated with 11-deoxycorticosterone (DOC), the 11beta-hydroxylation activity of the mutants was most effectively increased by combining D147E and I112P (sixfold increase). The two substitutions displayed an additive effect. The same tendency can be observed when using 11-deoxycortisol as a substrate, although the effect is less pronounced. The second step of the CYP11B2-dependent DOC conversion, the 18-hydroxylation activity, was not as strongly increased as the 11beta-hydroxylation potential. Activity was unaffected by D147E, whereas the single mutant I112P displayed the most pronounced activation (70% enhancement), thus causing different increasing effects on the first two enzymatic reaction steps. A slightly enhanced aldosterone synthesis from DOC could be measured due to increased levels of the intermediates. However, the 18-oxidation activity of all the mutants, except for I112S and D147E, was slightly reduced. The strongly enhanced 18-hydroxycorticosterone and aldosterone formation observed in the mutants provides important information on a possible role of such amino-acid replacements in the development of essential hypertension. Furthermore, the results indicate the possibility of a differential as well as independent modification of CYP11B2 reaction steps. The combination of functional data and computer modelling of CYP11B2 suggests an indirect involvement of residue 147 in the regulation of CYP11B isoform specific substrate conversion due to its location on the protein surface. In addition, the results indicate the functional significance of amino-acid 112 in the putative substrate access channel of human CYP11B2. Thus, we present

Low temperature resistivity studies of SmB6: Observation of two-dimensional variable-range hopping conductivity

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Batkova, Marianna; Batko, Ivan; Gabáni, Slavomír; Gažo, Emil; Konovalova, Elena; Filippov, Vladimir

2018-05-01

We studied electrical resistance of a single-crystalline SmB6 sample with a focus on the region of the "low-temperature resistivity plateau". Our observations did not show any true saturation of the electrical resistance at temperatures below 3 K down to 70 mK. According to our findings, temperature dependence of the electrical conduction in a certain temperature interval above 70 mK can be decomposed into a temperature-independent term and a temperature-activated term that can be described by variable-range hopping formula for two-dimensional systems, exp [ -(T0 / T) 1 / 3 ]. Thus, our results indicate importance of hopping type of electrical transport in the near-surface region of SmB6.

Bacterial Production and Enzymatic Activities in Deep-Sea Sediments of the Pacific Ocean: Biogeochemical Implications of Different Temperature Constraints

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Danovaro, R.; Corinaldesi, C.; dell'Anno, A.

2002-12-01

The deep-sea bed, acting as the ultimate sink for organic material derived from the upper oceans primary production, is now assumed to play a key role in biogeochemical cycling of organic matter on global scale. Early diagenesis of organic matter in marine sediments is dependent upon biological processes (largely mediated by bacterial activity) and by molecular diffusion. Organic matter reaching the sea floor by sedimentation is subjected to complex biogeochemical transformations that make organic matter largely unsuitable for direct utilization by benthic heterotrophs. Extracellular enzymatic activities in the sediment is generally recognized as the key step in the degradation and utilization of organic polymers by bacteria and a key role in biopolymeric carbon mobilization is played by aminopeptidase, alkaline phosphatase and glucosidase activities. In the present study we investigated bacterial density, bacterial C production and exo-enzymatic activities (aminopeptidase, glucosidase and phosphatase activity) in deep-sea sediments of the Pacific Ocean in relation with the biochemical composition of sediment organic matter (proteins, carbohydrates and lipids), in order to gather information on organic matter cycling and diagenesis. Benthic viral abundance was also measured to investigate the potential role of viruses on microbial loop functioning. Sediment samples were collected at eight stations (depth ranging from 2070-3100 m) along two transects located at the opposite side (north and south) of ocean seismic ridge Juan Fernandez (along latitudes 33° 20' - 33° 40'), constituted by the submerged vulcanoes, which connects the Chilean coasts to Rapa Nui Island. Since the northern and southern sides of this ridge apparently displayed small but significant differences in deep-sea temperature (related to the general ocean circulation), this sampling strategy allowed also investigating the role of different temperature constraints on bacterial activity and

Evaluation of physical structural features on influencing enzymatic hydrolysis efficiency of micronized wood

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Jinxue Jiang; Jinwu Wang; Xiao Zhang; Michael Wolcott

2016-01-01

Enzymatic hydrolysis of lignocellulosic biomass is highly dependent on the changes in structural features after pretreatment. Mechanical milling pretreatment is an effective approach to alter the physical structure of biomass and thus improve enzymatic hydrolysis. This study examined the influence of structural characteristics on the enzymatic hydrolysis of micronized...

The enzymatic activity of the VEGFR2 receptor for the biosynthesis of dinucleoside polyphosphates.

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Jankowski, Vera; Schulz, Anna; Kretschmer, Axel; Mischak, Harald; Boehringer, Falko; van der Giet, Markus; Janke, Doreen; Schuchardt, Mirjam; Herwig, Ralf; Zidek, Walter; Jankowski, Joachim

2013-09-01

The group of dinucleoside polyphosphates encompasses a large number of molecules consisting of two nucleosides which are connected by a phosphate chain of variable length. While the receptors activated by dinucleoside polyphosphates as well as their degradation have been studied in detail, its biosynthesis has not been elucidated so far. Since endothelial cells released the dinucleoside polyphosphate uridine adenosine tetraphosphate (Up4A), we tested cytosolic proteins of human endothelial cells obtained from dermal vessels elicited for enzymatic activity. When incubated with ADP and UDP, these cells showed increasing concentrations of Up4A. The underlying enzyme was isolated by chromatography and the mass spectrometric analysis revealed that the enzymatic activity was caused by the vascular endothelial growth factor receptor 2 (VEGFR2). Since VEGFR2 but neither VEGFR1 nor VEGFR3 were capable to synthesise dinucleoside polyphosphates, Tyr-1175 of VEGFR2 is most likely essential for the enzymatic activity of interest. Further, VEGFR2-containing cells like HepG2, THP-1 and RAW264.7 were capable of synthesising dinucleoside polyphosphates. VEGFR2-transfected HEK 293T/17 but not native HEK 293T/17 cells synthesised dinucleoside polyphosphates in vivo too. The simultaneous biosynthesis of dinucleoside polyphosphates could amplify the response to VEGF, since dinucleoside polyphosphates induce cellular growth via P2Y purinergic receptors. Thus the biosynthesis of dinucleoside polyphosphates by VEGFR2 may enhance the proliferative response to VEGF. Given that VEGFR2 is primarily expressed in endothelial cells, the biosynthesis of dinucleoside polyphosphates is mainly located in the vascular system. Since the vasculature is also the main site of action of dinucleoside polyphosphates, activating vascular purinoceptors, blood vessels appear as an autocrine system with respect to dinucleoside polyphosphates. We conclude that VEGFR2 receptor is capable of synthesising

Method for the enzymatic production of hydrogen

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Woodward, J.; Mattingly, S.M.

1999-08-24

The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

Site-Specific Bioconjugation of an Organometallic Electron Mediator to an Enzyme with Retained Photocatalytic Cofactor Regenerating Capacity and Enzymatic Activity

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Sung In Lim

2015-04-01

Full Text Available Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(PH being readily available to a redox enzyme, when the local NAD(PH concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH. A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

Energy Technology Data Exchange (ETDEWEB)

Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

2015-10-23

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis.

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Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H

2016-08-12

Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase. II. The influence of surface potential upon the activating ion equilibria.

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Ahrens, M L

1983-07-13

Electrostatic influences upon the enzymatic activity of the (Na+ + K+)-ATPase from ox brain (EC 3.6.1.3) have been studied. (1) The characteristics of the temperature dependence of the activity - the slopes and inflection temperature, Ti, of the Arrhenius plots - have been shown to depend on the total concentration, but not on the specific properties of added monovalent ions. (2) The enzymatic activity has been shown to be subject simultaneously to unspecific and specific influences of alkali-metal ions or NH+4. Ion-specific effects result from different binding constants of complexation between activating ions and enzyme. These stability constants are affected by the formation of an electrical double layer at the membrane surface. With increasing electrostatic screening, the complex formation is destabilized and, as a consequence, the enzymatic activity decreases. (3) This interaction between ion binding and surface electrostatics enables the enzyme to adapt its activity to the actual ionic conditions. This gives rise to a complex net dependence of the enzymatic activity upon the concentrations of activating ions. Such dependencies are analyzed, and an 'activity surface' has been constructed which represents the enzymatic activity as a function of simultaneously varying concentrations of sodium and potassium. The shape of this activity surface is determined by the relations between ion concentrations, surface potential and the resulting stability of the complexation between the activating ions and the enzyme. By means of three-dimensional representation it is demonstrated that the adaptability of the stability constants is of great importance with respect to the maintenance of the optimal ionic concentrations within the living cell. Therefore, by means of the surrounding membrane, the ATPase is provided with a quality, in addition to its substrate specificity and catalytic ability, which is necessary for its function as a transport enzyme.

Aespoe Task Force on modelling of groundwater flow and transport of solutes. Review of Tasks 6b, 6b and 6b

Energy Technology Data Exchange (ETDEWEB)

Hodgkinson, David [Quintessa, Henley-on-Thames (United Kingdom); Black, John [In Situ Solutions, East Bridgford (United Kingdom)

2005-03-01

This report forms part of an independent review of the specifications, execution and results of Task 6 of the Aespoe Task Force on Modelling of Groundwater Flow and Transport of Solutes, which is seeking to provide a bridge between site characterization and performance assessment approaches to solute transport in fractured rock. The present report is concerned solely with Tasks 6b, 6b and 6b which relate to the transport of tracers on a 5-metre scale in Feature A at the TRUE-1 site. The task objectives, specifications and individual modelling team results are summarised and reviewed, and an evaluation of the overall exercise is presented. The report concludes with assessments of what has been learnt, the implications for the Task 6 objectives, and some possible future directions.

Methods for determining enzymatic activity comprising heating and agitation of closed volumes

Science.gov (United States)

Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

2016-03-15

Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system.

Science.gov (United States)

Zeng, Lu S; Liao, Min; Chen, Cheng L; Huang, Chang Y

2007-05-01

The effect of lead (Pb) treatment on the soil enzymatic activities, soil microbial biomass, rice physiological indices and rice biomass were studied in a greenhouse pot experiment. Six levels of Pb viz. 0(CK), 100, 300, 500, 700, 900 mg/kg soil were applied in two types of paddy soils. The results showed that Pb treatment had a stimulating effect on soil enzymatic activities and microbial biomass carbon (Cmic) at low concentration and an inhibitory influence at higher concentration. The degree of influence on enzymatic activities and Cmic by Pb was related to the clay and organic matter contents of the soils. When the Pb treatment was raised to the level of 500 mg/kg, ecological risk appeared both to soil microorganisms and plants. The results also revealed a consistent trend of increased chlorophyll contents and rice biomass initially, maximum at a certain Pb treatment, and then decreased gradually with the increase in Pb concentration. Pb was effective in inducing proline accumulation and its toxicity causes oxidative stress in rice plants. Therefore, it was concluded that soil enzymatic activities, Cmic and rice physiological indices, could be sensitive indicators to reflect environmental stress in soil-lead-rice system.

Increased p50/p50 NF-κB Activation in Human Papillomavirus Type 6- or Type 11-Induced Laryngeal Papilloma Tissue

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Vancurova, Ivana; Wu, Rong; Miskolci, Veronika; Sun, Shishinn

2002-01-01

We have observed elevated NF-κB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-κB activity could be correlated with a reduced level of cytoplasmic IκBβ and could be associated with the overexpression of p21CIP1/WAF1 in papilloma cells. Increased NF-κB activity and cytoplasmic accumulation of p21CIP1/WAF1 might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues. PMID:11773428