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Sample records for extracellular enzyme activity

  1. Detection of Extracellular Enzyme Activities in Ganoderma neo-japonicum

    OpenAIRE

    Jo, Woo-Sik; Park, Ha-Na; Cho, Doo-Hyun; Yoo, Young-Bok; Park, Seung-Chun

    2011-01-01

    The ability of Ganoderma to produce extracellular enzymes, including β-glucosidase, cellulase, avicelase, pectinase, xylanase, protease, amylase, and ligninase was tested in chromogenic media. β-glucosidase showed the highest activity, among the eight tested enzymes. In particular, Ganoderma neo-japonicum showed significantly stronger activity for β-glucosidase than that of the other enzymes. Two Ganoderma lucidum isolates showed moderate activity for avicelase; however, Ganoderma neo-japonic...

  2. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    and soil ecosystem services. The soil enzyme activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysaccharides as cellulose, hemicellulose and chitin, while degradation of proteins has been......Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soil enzymes originate from a variety of organisms, notably fungi and bacteria......, experimental conditions of extraction of enzymes from soils, buffer and pH, substrate concentration, temperature and the necessary controls were optimized and standardized. This has resulted in an optimized standard operating procedure of EEA, which are being tested as an indicator of soil functional diversity...

  3. Understanding drivers of peatland extracellular enzyme activity in the PEATcosm experiment: mixed evidence for enzymic latch hypothesis

    Science.gov (United States)

    Karl J. Romanowicz; Evan S. Kane; Lynette R. Potvin; Aleta L. Daniels; Randy Kolka; Erik A. Lilleskov

    2015-01-01

    Aims. Our objective was to assess the impacts of water table position and plant functional groups on peatland extracellular enzyme activity (EEA) framed within the context of the enzymic latch hypothesis. Methods. We utilized a full factorial experiment with 2 water table (WT) treatments (high and low) and 3 plant functional...

  4. Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

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    Yumiko Obayashi

    2017-10-01

    Full Text Available Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO and 2-methoxyethanol (MTXE. The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube, protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In

  5. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... awareness of the threats to soil and the ecosystem services has fostered the need for a thorough understanding of soil functions and activities. Soils are very species-rich and show great functional heterogeneity. While molecular analysis of gene expression is developing, simple and inexpensive indicators......, recycling of nutrients and waste, soil remediation, plant growth support and regulation of above ground biodiversity, resilience, and soil suppressiveness. As such, soil ecosystem services are beneficial and vital for human life and at the same time threatened by anthropogenic activities. Increasing...

  6. Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

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    Elisabeth Pohlon

    Full Text Available Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany. Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes

  7. Soil Minerals Affect Extracellular Enzyme Activities in Cold and Warm Environments

    Science.gov (United States)

    Yang, Z.; Morin, M. M.; Graham, D. E.; Wullschleger, S. D.; Gu, B.

    2017-12-01

    Extracellular enzymes are mainly responsible for degrading and cycling soil organic matter (SOM) in both cold and warm terrestrial ecosystems. Minerals can play important roles in affecting soil enzyme activities, however, the interactions between enzyme and soil minerals remain poorly understood. In this study, we developed a model soil-enzyme system to examine the mineral effects on a hydrolytic enzyme (i.e., β-glucosidase) under both cold (4°C) and relatively warm (20 and 30°C) conditions. Minerals including iron oxides and clays (e.g., kaolinite and montmorillonite) were used to mimic different types of soils, and enzyme adsorption experiments were conducted to determine the enzyme interactions with different mineral surfaces. Time-series experiments were also carried out to measure enzymatic degradation of the organic substrates, such as cellobiose and indican. We observed that fractions of adsorbed enzyme and the hydrolytic activity were higher on iron oxides (e.g., hematite) compared to kaolinite and montmorillonite at given experimental conditions. The degradation of cellobiose was significantly faster than that of indican in the presence of minerals. We also found that the adsorption of enzyme was not dependent on the mineral surface areas, but was controlled by the mineral surface charge. In addition, temperature increase from 4 to 30°C enhanced mineral-assisted glucosidase hydrolysis by 2 to 4 fold, suggesting greater degradation under warmer environments. The present work demonstrates that the enzyme activity is influenced not only by the soil temperature but also by the surface chemistry of soil minerals. Our results highlight the need to consider the physical and chemical properties of minerals in biogeochemical models, which could provide a better prediction for enzyme-facilitated SOM transformations in terrestrial ecosystems.

  8. Stimulation of microbial extracellular enzyme activities by elevated CO2 depends on soil aggregate size

    Science.gov (United States)

    Dorodnikov, M.; Blagodatskaya, E.; Blagodatsky, S.; Marhan, S.; Fangmeier, A.; Kuzyakov, Y.

    2009-04-01

    Increased belowground carbon (C) transfer by plant roots at elevated CO2 may change properties of the microbial community in the rhizosphere. Previous investigations that focused on total soil organic C or total microbial C showed contrasting results: small increase, small decrease or no changes. We evaluated the effect of 5 years of elevated CO2(550 ppm) on four extracellular enzymes: ß-glucosidase, chitinase, phosphatase, and sulfatase. We expected microorganisms to be differently localized in aggregates of various sizes and, therefore analyzed microbial biomass (Cmic by SIR) and enzyme activities in three aggregate-size classes: large macro- (>2 mm), small macro- (0.25-2 mm), and microaggregates (production, we activated microorganisms by substrate (glucose and nutrients) amendment. Although Ctotal and Cmic as well as the activities of ß-glucosidase, phosphatase, and sulfatase were unaffected in bulk soil and in aggregate-size classes by elevated CO2, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. This indicates the increased activity of microorganisms, which leads to accelerated C turnover in soil under elevated CO2. Significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. At the same time, less chitinase activity in microaggregates underlined microaggregate stability and the difficulties for fungal hyphae penetrating them. We conclude that quantitative and qualitative changes of C input by plants into the soil at elevated CO2 affect microbial community functioning, but not its total content. Future studies should therefore focus more on the changes of functions and activities, but less on the pools.

  9. Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

    Science.gov (United States)

    Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2014-05-01

    detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

  10. Evidence for extracellular enzymic activity of the isolated perfused rat heart

    Science.gov (United States)

    Williamson, John R.; DiPietro, David L.

    1965-01-01

    1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of 14CO2 from [U-14C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-14C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-14C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane. PMID:14333561

  11. Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

    Science.gov (United States)

    Brzostek, E. R.; Phillips, R.; Dragoni, D.; Drake, J. E.; Finzi, A. C.

    2011-12-01

    The mobilization of nitrogen (N) from soil organic matter in temperate forest soils is controlled by the microbial production and activity of extracellular enzymes. The exudation of carbon (C) by tree roots into the rhizosphere may subsidize the microbial production of extracellular enzymes in the rhizosphere and increase the access of roots to N. The objective of this research was to investigate whether rates of root exudation and the resulting stimulation of extracellular enzyme activity in the rhizosphere (i.e., rhizosphere effect) differs between tree species that form associations with ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. This research was conducted at two temperate forest sites, the Harvard Forest (HF) in Central MA and the Morgan Monroe State Forest (MMSF) in Southern IN. At the HF, we measured rates of root exudation and the rhizosphere effects on enzyme activity, N cycling, and C mineralization in AM and ECM soils. At the MMSF, we recently girdled AM and ECM dominated plots to examine the impact of severing belowground C allocation on rhizosphere processes. At both sites, the rhizosphere effect on proteolytic, chitinolytic and ligninolytic enzyme activities was greater in ECM soils than in AM soils. In particular, higher rates of proteolytic enzyme activity increased the availability of amino acid-N in ECM rhizospheres relative to the bulk soils. Further, this stimulation of enzyme activity was directly correlated with higher rates of C mineralization in the rhizosphere than in the bulk soil. Although not significantly different between species, root exudation of C comprised 3-10% of annual gross primary production at the HF. At the MMSF, experimental girdling led to a larger decline in soil respiration and enzyme activity in ECM plots than in AM plots. In both ECM and AM soils, however, girdling resulted in equivalent rates of enzyme activity in rhizosphere and corresponding bulk soils. The results of this study contribute to the

  12. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  13. Effect of extracellular enzyme activity on digestion performance of mesophilic UASB reactor treating high-strength municipal wastewater.

    Science.gov (United States)

    Turkdogan-Aydinol, F Ilter; Yetilmezsoy, Kaan; Comez, Sezen

    2011-05-01

    Effect of extracellular enzyme activity on digestion performance of up-flow anaerobic sludge blanket (UASB) reactor was investigated for enhancement of anaerobic treatability of municipal wastewater. Two identical UASB reactors (9 L), namely Reactor-A (without enzyme addition) and Reactor-B (with enzyme addition),were simultaneously operated at mesophilic conditions (32 ± 2°C) with a hydraulic retention time of 24 h. Preliminary test results showed that the highest total chemical oxygen demand (TCOD) removal were achieved with an extracellular enzyme dosage of 0.2 mL/L. In the activation period of the extracellular enzyme (on days 186-212), while Reactor-A removed up to 69.3% of TCOD and 55.9% of soluble chemical oxygen demand (SCOD), Reactor-B effectively removed up to 81.9% of TCOD and 72.2% of SCOD. The average VFA/alkalinity ratios were determined to be about 0.40 (±0.03) and 0.28 (±0.08) for Reactor-A and Reactor-B, respectively.

  14. Primary effects of extracellular enzyme activity and microbial community on carbon and nitrogen mineralization in estuarine and tidal wetlands.

    Science.gov (United States)

    Li, Xiaofei; Hou, Lijun; Liu, Min; Lin, Xianbiao; Li, Ye; Li, Shuwen

    2015-03-01

    Estuarine and tidal wetlands with high primary productivity and biological activity play a crucial role in coastal nutrient dynamics. Here, to better reveal the effects of extracellular enzymes and microbial community on carbon (C) and nitrogen (N) mineralization, the incubation experiments with different C and N addition patterns to the tidal sediments of the Yangtze Estuary (China) were conducted. The results suggested a significant increase in cumulative CO2 effluxes in the C and CN treatment experiments, while no significant difference in cumulative CO2 effluxes between the N treatment and control (CK) experiments was observed. In addition, the nutrient addition patterns had a great influence on dissolve organic C and N levels, but a small effect on microbial biomass C and N. Microbial community composition and microbial activity were found to be positively correlated with organic C (OC) and the molar ratio of C to N (C/N). Partial correlation analysis, controlling for C/N, supported direct effects of OC on the activity of carbon-cycling extracellular enzymes (cellulase and polyphenol oxidase), while C/N exhibited negatively correlations with urease and Gram-positive bacteria to Gram-negative bacteria (G+/G-). Strong relationships were found between CO2 efflux and mineral nitrogen with the activity of specific enzymes (sucrase, cellulase, and polyphenol oxidase) and abundances of Gram-negative bacteria, arbuscular mycorrhizal fungi, and fungi, suggesting the significant influences of microbial community and enzyme activity on C and N mineralization in the estuarine and tidal wetlands. Furthermore, this study could highlight the need to explore effects of nutrient supply on microbial communities and enzyme activity changes associated with the C and N mineralization in these wetlands induced by the climate change.

  15. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

    International Nuclear Information System (INIS)

    Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

    1987-01-01

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

  16. Extracellular enzyme activities of aquatic bacteria in polluted environment: 2. Amylolytic activity

    International Nuclear Information System (INIS)

    Arbaciauskiene, V.

    2003-01-01

    Water samples were taken from Lake Drukshiai tributaries (Ricanka); Gulbinele Stream affected by urban rain sewerage from Visaginas; Gulbinele Stream into which municipal sewage from Visaginas (MS) and industrial rain sewerage from the Ignalina NPP and their mouth, and Lake Dringis. Lake Dringis, in Aukstaitija National Park, was selected as an ecosystem pattern of a weak anthropogenic influence, while Lake Drukshiai was chosen as a regularly polluted water body. Lake Drukshiai, the cooling basin of the Ignalina NPP (IRS-1.2), is being polluted with industrial and municipal sewage through its tributaries. The amylolytic activity (AA) of heterotrophic aquatic bacteria was tested. The highest total mean AA of aquatic bacteria was calculated in Lake Dringis. Here, the results were significantly higher than in Lake Drukshiai tributaries and their mouths, excepting the mouths of the Ricanka and MS. The lowest mean of AA in Lake Drukshiai was characteristic of the IRS-1.2 tributary. A comparison of the mean AA of active isolates showed that certain bacterial strains from the sites of varying degrees of pollution could be noted for a relatively high level of enzymatic activity. Thus, anthropogenic pollution exerts a negative effect on the total mean AA, although certain strains of bacteria are able to adapt to the stressful environment and remain active. (author)

  17. Indication of temperature inverted microbial assimilative capacities (extracellular enzymes activities in the pelagic of Lake Sevan (Armenia

    Directory of Open Access Journals (Sweden)

    Arevik MINASYAN

    2016-06-01

    Full Text Available Pioneering records of extracellular enzymes activities (EEA in Lake Sevan waters highlight dependence of heterotrophic functioning on physicochemical characteristics and bacterial assemblage. Values of EEA, ranged 0.11-30.39 µg C/P L-1h-1, were higher in upper layers compared to the omission in deeper parts. Particles associated (ecto- enzymes mainly predominated over free dissolved (exo- enzymes. In June activities of all studied enzymes followed similar pattern, particularly, decreasing at thermocline and increasing twice/more in cold deeper waters. Regardless higher bacterial density and temperature in June, with no similar records up to now, EEA revealed reverse relationship to temperature and bacteria data and were significantly lesser than in March. Our finding might be suggested as temperature inverted impact to heterotrophic activities in eutrophic conditions. We assume that observed, with temperature raise, declined EEA was due to blocked enzymatic active center from colloids and DOM components interaction, which, in overall, may suppress organic substrate utilization and result in weakening of first and rate limiting step of biological self-purification in Lake Sevan waters. Therefore, since temperature is co-regulator of assimilative/carrying capacity of aquatic ecosystems, climate warming might have unexpected negative feedbacks also through lowering assimilative capacities of water bodies, jeopardizing their quality and ecology.

  18. Extracellular enzymes of Fusarium graminearum isolates

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    Gisele Eleonora Kikot

    2010-08-01

    Full Text Available Fusarium graminearum isolates from three different agroecological regions in Argentina were examined according to the production of different extracellular enzyme activities of potential biotechnological interest: pectinases (PGase: polygalacturonase and PMGase: polymethylgalacturonase, cellulase (CMCase: carboxymethylcellulase and hemicellulase (xylanase. The isolates were grown in minimum salt medium supplemented with 0.25% glucose, 0.125% citric pectin and 0.125% oat bran as carbon sources and/or enzyme inducers. PGase activity was detected early (after two days of incubation in all the cultures; it was found to be the highest for all the isolates. PMGase was high only for those isolates of the II region. CMCase and endoxylanase activities were particularly found at late stages (after four and seven days of incubation, respectively and the maximum values were lower than pectinase activities.

  19. In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

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    Arumugam Mythili

    2014-10-01

    Full Text Available AIM:To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates.METHODS:The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation. Extracellular enzymes such as lipase, deoxyribonuclease (DNase, α-amylase, protease, cellulase and pectinase produced by the fungalisolates were screened on solid media supplemented with the corresponding substrates. Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungalisolates.RESULTS:A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these, Fusarium spp. (n=30, non sporulating molds (n=9, Aspergillus flavus (n=6, Bipolaris spp. (n=6, Exserohilum spp. (n=4, Curvularia spp. (n=3, Alternaria spp. (n=1 and Exophiala spp. (n=1were identified and designated as FS1-30, NSM1-9, AF1-6, BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp., Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2 (EAI= 2.14. The DNase activity was higher in NSM9 (EAI=1.88. In case of protease, Fusarium spp. (FS9 had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i

  20. High activity and low temperature optima of extracellular enzymes in Arctic sediments: implications for carbon cycling by heterotrophic microbial communities

    DEFF Research Database (Denmark)

    Arnosti, C.; Jørgensen, BB

    2003-01-01

    The rate of the initial step in microbial remineralization of organic carbon, extracellular enzymatic hydrolysis, was investigated as a function of temperature in permanently cold sediments from 2 fjords on the west coast of Svalbard (Arctic Ocean). We used 4 structurally distinct polysaccharides...... hydrolysis in order to determine the relative temperature responses of the initial and terminal steps in microbial remineralization of carbon. The temperature optimum of sulfate reduction, 21degreesC, was considerably lower than previous reports of sulfate reduction in marine sediments, but is consistent...... with recent studies of psychrophilic sulfate reducers isolated from Svalbard sediments. A calculation of potential carbon flow into the microbial food chain demonstrated that the activity of just one type of polysaccharide-hydrolyzing enzyme could in theory supply 21 to 100% of the carbon consumed via sulfate...

  1. Differentiation between activity of digestive enzymes of .i.Brachionus calyciflorus./i. and extracellular enzymes of its epizooic bacteria

    Czech Academy of Sciences Publication Activity Database

    Štrojsová, Martina; Ahlrichs, W.H.

    2009-01-01

    Roč. 68, č. 2 (2009), s. 409-412 ISSN 1129-5767 Institutional research plan: CEZ:AV0Z60170517 Keywords : rotifers * phosphatase * beta-N-acetylhexosaminidase * enzyme localization Subject RIV: EG - Zoology Impact factor: 0.932, year: 2009

  2. Patterns of extracellular enzyme activities in littoral sediments of Lake Gooimeer, The Netherlands

    NARCIS (Netherlands)

    Boschker, H.T.S.; Cappenberg, T.E.

    1998-01-01

    The seasonal variation in enzymatic activities against protein and several polysaccharides together with esterase as a measure of general hydrolytic activity was studied in a transect of sediments through the littoral zone of Lake Gooimeer, The Netherlands. Absolute activities showed a linear

  3. Early-successional ectomycorrhizal fungi effectively support extracellular enzyme activities and seedling nitrogen accumulation in mature forests.

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    Nicholson, Bailey A; Jones, Melanie D

    2017-04-01

    After stand-replacing disturbance, regenerating conifer seedlings become colonized by different ectomycorrhizal fungi (EMF) than the locally adapted EMF communities present on seedlings in mature forests. We studied whether EMF species that colonized subalpine fir (Abies lasiocarpa) seedlings in clearcuts differed from those that colonized seedlings in adjacent mature forests with respect to mycorrhizoplane extracellular enzyme activities (EEAs) and N status of the seedlings. We tested two alternate hypotheses: (1) that EEAs would differ between the two EMF communities, with higher activities associated with forest-origin communities, and (2) that acclimation to soil environment was considerable enough that EEAs would be determined primarily by the soil type in which the ectomycorrhizas were growing. Naturally colonized fir seedlings were reciprocally transplanted between clearcuts and forests, carrying different EMF communities with them. EEAs were influenced more by destination environment than by EMF community. EEAs were as high in early-successional as in late-successional communities in both destination environments. Buds of clearcut-origin seedlings had the same or higher N contents as forest seedlings after a growing season in either environment. These results indicate that (i) symbiotic EMF and/or their associated microbial communities demonstrate substantial ability to acclimate to new field environments; (ii) the ability to produce organic matter-degrading enzymes is not a trait that necessarily distinguishes early- and late-successional EMF communities in symbiosis; (iii) early-successional EMF are as capable of supporting seedling N accumulation in forest soils as late-successional EMF; and (iv) disturbed ecosystems where early-successional EMF are present should have high resilience for organic matter degradation.

  4. Extracellular hydrolytic enzyme activities of the heterotrophic microbial communities of the Rouge River: an approach to evaluate ecosystem response to urbanization.

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    Tiquia, S M

    2011-10-01

    The potential effects of urbanization on the bioavailability of dissolved organic carbon (DOC) were tested by determining the extracellular enzyme activities of the heterotrophic microbial communities of the Rouge River. The activities of 19 enzymes were monitored across two water samples (river water and groundwater) at different spatial and temporal scales. High phosphatase, esterase, and aminopeptidase activities was observed in site 9 (site most exposed to anthropogenic sources) showed higher concentrations of DOC compared to sites 1 and 8 (sites exposed to less anthropogenic sources), where moderate activities of diverse range of enzymes were observed. High relative contributions of phosphatase, esterase, and aminopeptidase activities to the overall enzyme activity as observed in site 9 stressed the increased importance of peptides as C source for heterotrophic communities and high in-stream carbon processing, which account for high nonspecific extracellular enzyme activities. In contrast, high contribution of glycosyl hydrolases occurred consistently across all sites, which highlights the significance of microbial detrital and plant biomass as carbon sources. Majority of the enzymes showed evidence of activity at various extents during spring and summer. However, higher activities of leucine aminopeptidase, valine aminopeptidase, β-glucosidase, and α-mannosidase were observed in the summer; and alkaline phosphatase and α-glucosidase in the spring. The results presented here suggest a shift in organic carbon bioavailability across all sites of contrasting urbanization, despite similarities in DOC concentrations. Hence, API ZYM technique can be used as an effective indicator of river water and groundwater system health across an urban gradient.

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

    Science.gov (United States)

    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).

  6. Characterization and Extracellular Enzyme Activity of Predominant Marine Bacillus spp. Isolated From Sea Water of Orissa Coast, India

    Directory of Open Access Journals (Sweden)

    Bal, S.

    2009-01-01

    Full Text Available Bacillus species are ubiquitous and diverse both in the terrestrial and marine ecosystems. In this investigation, predominant Bacillus species from sea water of three different sites of Orissa Coast were isolated and identified. In total, 16 Bacillus species were identified using morpho-physiological and biochemical characterisation. These identified bacterial strains include B. fastidiosus (CMB1, B. alvei (CMB2, B. coagulans (CMB3, B. marinus (CMB5, B. mycoides (CMB8, B. coagulans (PMB1, B. circulans (PMB2, B. cereus (PMB3, B. subtilis (PMB4, B. alcalophilus (GMB1, B. licheniformics (GMB2, B. polymyxa (GMB3 and B. pumilus (GMB4. The isolates CMB4, CMB6 and CMB7 were identified only up to genus level. These isolates were further screened for their salt tolerance and growth under varied temperature and pH conditions. Ability of these strains to produce extracellular enzymes such as protease, amylase, lipase, gelatinase, casein hydrolase, lecithinase, chitinase and pectinase were also screened and found that most of the Bacillus spp. possess extracellular enzymes.

  7. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    In the present study, the amylase enzyme producing potential of four different Aspergillus species was analyzed. The extracted amylase enzyme was purified by diethyl amino ethyl (DEAE) cellulose and Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch.

  8. Growth and extracellular enzyme production by microorganisms ...

    African Journals Online (AJOL)

    Ugba', an indigenous Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined. The effect of pH and ...

  9. growth and extracellular enzyme production by microorganisms

    African Journals Online (AJOL)

    Okorie

    2013-06-26

    Ugba', an indigenous. Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined.

  10. Production of multiple extracellular enzyme activities by novel submerged culture of Aspergillus kawachii for ethanol production from raw cassava flour.

    Science.gov (United States)

    Sugimoto, Toshikazu; Makita, Tomohiro; Watanabe, Koutaro; Shoji, Hiroshi

    2012-04-01

    Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.

  11. Effects of dissolved oxygen on extracellular enzymes activities and transformation of carbon sources from plant biomass: implications for denitrification in constructed wetlands.

    Science.gov (United States)

    Chen, Yi; Wen, Yue; Cheng, Jing; Xue, ChongHua; Yang, Dianhai; Zhou, Qi

    2011-02-01

    Dissolved oxygen (DO) concentrations have often been shown to be important to decomposition rates of plant litter and thus may be a key factor in determining the supply of dissolved organic carbon (DOC) and carbon-dependent denitrification in wetlands. During the 2 months operation, DOC accumulation in anaerobic condition was superior to aerobic condition due to higher activities of hydrolase enzymes and lower hydrolysates converted to gaseous C. Also, much higher denitrification rates were observed in wetland when using anaerobic litter leachate as the carbon source, and the available carbon source (ACS) could be used as a good predictor of denitrification rate in wetland. According to the results of this study, extracellular enzymes activities (EEAs) in wetland would change as a short-term consequence of DO. This may alter balance of litter carbon flux and the characteristics of DOC, which may, in turn, have multiple effects on denitrification in wetlands. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

    Science.gov (United States)

    Luo, Ling; Meng, Han; Gu, Ji-Dong

    2017-07-15

    Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Characterization of Extracellular Chitinolytic Activity in Biofilms

    Energy Technology Data Exchange (ETDEWEB)

    Baty, Ace M.; Diwu, Zhenjun; Dunham, Glen C.; Eastburn, Callie; Geesey, Gill G.; Goodman, Amanda; Suci, Peter; Techkarnjanaruk, Somkiet

    2001-05-01

    It is common for bacteria to produce extracellular enzymes having some form of degradative activity. In some cases these enzymes serve to protect cells from antagonistic substances, or to convert a large and/or insoluble biopolymer to an assimilable nutrient source. In some cases the physiological benefit to the bacterium is not entirely evident. Extracellular enzymes may be membrane bound, but in many cases they are released into the surrounding medium. It has been shown that these relatively large molecules become immobilized in the extracellular polymeric matrix in which cells in flocs and biofilms are embedded. Most proteins adsorb irreversibly to substrata having a variety of surface chemistries, and transport by convection is reduced near any solid surface, regardless of the flow regimen in the bulk liquid. Thus, extracellular enzymes have a tendency to become an integral and significant component of the biofilm/substratum microenvironment, influencing cell physiology and biofilm ecology.

  14. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    Science.gov (United States)

    Zhou, Xiaoqi; Wang, Shen S. J.; Chen, Chengrong

    2017-12-01

    Forest plantations have been widely used as an effective measure for increasing soil carbon (C), and nitrogen (N) stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA) to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species-enzyme-C/N model to investigate how temperature and tree species influence soil C/N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG), N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP) and phosphorus acquisition enzymes (acid phosphatases). The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01-2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99-2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii) and hoop pine (Araucaria cunninghamii Ait.), increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22-1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native coniferous tree species like hoop pine and

  15. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    Directory of Open Access Journals (Sweden)

    X. Zhou

    2017-12-01

    Full Text Available Forest plantations have been widely used as an effective measure for increasing soil carbon (C, and nitrogen (N stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species–enzyme–C∕N model to investigate how temperature and tree species influence soil C∕N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG, N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP and phosphorus acquisition enzymes (acid phosphatases. The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01–2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99–2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii and hoop pine (Araucaria cunninghamii Ait., increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22–1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native

  16. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    DOSS

    2012-10-16

    Oct 16, 2012 ... Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch. ... Key words: Aspergillus species, Sephadex G-50, column chromatography, diethyl amino ethyl (DEAE) cellulose. ... chemical properties characterized (Ravan et al., 2009). Although ...

  17. Interleukin-1beta regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells.

    Science.gov (United States)

    Zitta, Karina; Brandt, Berenice; Wuensch, Annegret; Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens; Albrecht, Martin

    2010-09-03

    After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1beta is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1beta on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1beta. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1beta resulted in a dose-dependent increase of cell proliferation (Ppig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1beta (Pheart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1beta as a key molecule guiding tissue remodelling events after myocardial infarction. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Properties and stabilization of an extracellular alpha-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 degrees C.

    Science.gov (United States)

    Piller, K; Daniel, R M; Petach, H H

    1996-01-04

    An extracellular alpha-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4%. It is a monomer with a molecular mass of about 60 kDa and a pI around 5. At 98 degrees C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of 1% (w/v) dithiothreitol and 1% (w/v) BSA. Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity. The Km at 75 degrees C is 0.41 mM with pNP-alpha-D-glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-alpha-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose. A variety of techniques including immobolization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100 degrees C. The half-life could be increased from about 4 min at 100 degrees C to 30-60 min at 130 degrees C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BSA, and by cross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130 degrees C.

  19. Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    International Nuclear Information System (INIS)

    Zitta, Karina; Brandt, Berenice; Wuensch, Annegret; Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens; Albrecht, Martin

    2010-01-01

    Research highlights: → Levels of IL-1β are increased in the pig myocardium after infarction. → Cultured pig heart cells possess IL-1 receptors. → IL-1β increases cell proliferation of pig heart cells in-vitro. → IL-1β increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. → IL-1β may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One

  20. Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    Energy Technology Data Exchange (ETDEWEB)

    Zitta, Karina; Brandt, Berenice [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Wuensch, Annegret [Institute of Molecular Animal Breeding and Biotechnology, Ludwig Maximilians University, Munich (Germany); Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Albrecht, Martin, E-mail: Albrecht@anaesthesie.uni-kiel.de [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany)

    2010-09-03

    Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined

  1. Responses of the extracellular enzyme activities in hardwood forest to soil temperature and seasonality and the potential effects of climate change

    Czech Academy of Sciences Publication Activity Database

    Baldrian, Petr; Šnajdr, Jaroslav; Merhautová, Věra; Dobiášová, Petra; Cajthaml, Tomáš; Valášková, Vendula

    2013-01-01

    Roč. 56, JAN 2013 (2013), s. 60-68 ISSN 0038-0717 R&D Projects: GA MŠk(CZ) LA10001; GA MŠk(CZ) ME10152; GA MZe QH72216 Institutional support: RVO:61388971 Keywords : Extracellular enzymes * Forest soil * Lignocellulose Subject RIV: EE - Microbiology , Virology Impact factor: 4.410, year: 2013

  2. Response of microbial extracellular enzyme activities and r- vs. K- selected microorganisms to elevated atmospheric CO2 depends on soil aggregate size

    Science.gov (United States)

    Dorodnikov, Maxim; Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

    2014-05-01

    Increased belowground carbon (C) transfer by plant roots under elevated atmospheric CO2 and the contrasting environment in soil macro- and microaggregates could affect properties of the microbial community in the rhizosphere. We evaluated the effect of 5 years of elevated CO2 (550 ppm) on four extracellular enzymes: ß-glucosidase, chitinase, phosphatase, and sulfatase along with the contribution of fast- (r-strategists) and slow-growing microorganisms (K-strategists) in soil aggregates. We fractionated the bulk soil from the ambient and elevated CO2 treatments of FACE-Hohenheim (Stuttgart) into large macro- (>2 mm), small macro- (0.25-2.00 mm), and microaggregates (production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. In addition, µ values were significantly higher under elevated than ambient CO2 for bulk soil, small macroaggregates, and microaggregates. Based on changes in µ, GMB, and lag-period, we conclude that elevated atmospheric CO2 stimulated the r-selected microorganisms, especially in soil microaggregates. In contrast, significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. We conclude that quantitative and qualitative changes of C input by plants into the soil at elevated CO2 affect microbial community functioning, but not its total content. An increase in r-selected microorganisms could accelerate C turnover in terrestrial ecosystems under a future CO2-elevated atmosphere.

  3. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    ChithrashreeGS

    2012-08-23

    Aug 23, 2012 ... In the present work, we studied the role of antioxidant scavenging enzymes of plant pathogenic bacteria: catalase ... Key words: Paddy, plant pathogenic bacteria, antioxidant scavenging enzymes, exopolysaccharide, virulence, ..... the development of a non-host hypersensitive reaction in lettuce. Plant ...

  4. Role of antioxidant scavenging enzymes and extracellular ...

    African Journals Online (AJOL)

    In the present work, we studied the role of antioxidant scavenging enzymes of plant pathogenic bacteria: catalase, ascorbate peroxidase and a virulence factor; extracelluar polysaccharide production in determining the virulence of Xanthomonas oryzae pv. oryzae (Xoo) isolates and its differential reaction to rice cultivars.

  5. Production Of Extracellular Enzymes By Some Soil Yeasts

    OpenAIRE

    Falih, A. M. [عبد الله مساعد خلف الفالح

    1997-01-01

    This study investigated the ability of soil yeasts, Geotrichum candidum, Geotrichum capitatum and Williopsis californica to produce extracellular enzymes (amylase, cellulase and protease) in vitro compared with that of a laboratory strain of Saccharomyces cerevisiae. It appears that the soil yeasts studied here were less amylolytic yeasts except the yeast G. candidum, which was highly effective at extracellular amylase production. The soil yeast W. californica was an average producer of cellu...

  6. Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

    Science.gov (United States)

    Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

    2016-01-01

    Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Production of extracellular proteolytic enzymes by Beauveria bassiana

    Directory of Open Access Journals (Sweden)

    Józefa Chrzanowska

    2014-08-01

    Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

  8. Degrading capability and activity of extracellular xylanase secreted ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-04-26

    Apr 26, 2012 ... crude enzyme, and the weight loss of hemicellulose of corn stalk core and rice straw was 84.7 and. 27.8%, respectively. Qualitative .... activity was defined as the amount of enzyme required to liberate 1 μg of xylose or glucose in 1 min from ... Effect of temperature on extracellular xylanase activity stability.

  9. ESTIMATION OF EXTRACELLULAR LIPOLYTIC ENZYME ACTIVITY BY THERMOPHILIC BACILLUS SP. ISOLATED FROM ARID AND SEMI-ARID REGION OF RAJASTHAN, INDIA

    Directory of Open Access Journals (Sweden)

    Deeksha Gaur

    2012-10-01

    Full Text Available Thermophilic organisms can be defined as, micro-organisms which are adapted to survive at high temperatures. The enzymes secreted by thermophilic bacteria are capable of catalyzing biochemical reactions at high temperatures. Thermophilic bacteria are able to produce thermostable lipolytic enzymes (capable of degradation of lipid at temperatures higher than mesophilic bacteria. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are quite beneficial in terms of discovering thermostable lipase enzymes. Due to great temperature fluctuation in hot arid and semi-arid region of Rajasthan, this area could serve as a good source for new thermophilic lipase producing bacteria with novel industrially important properties. The main objective of this research is the isolation and estimation of industrially important thermophilic lipase enzyme produced by thermophilic bacteria, isolated from arid and semi-arid region of Rajasthan. For this research purpose soil samples were collected from Churu, Sikar and Jhunjunu regions of Rajasthan. Total 16 bacterial strains were isolated and among only 2 thermostable lipolytic enzyme producing bacteria were charcterized. The thermostable lipolytic enzyme was estimated by qualitative and quantitative experiments. The isolates were identified as Bacillus sp. by microscopic, biochemical and molecular characterization. The optimum enzyme activity was observed at pH 8, temperature 60°C and 6% salt concentrations at 24 hrs time duration. Lipolytic enzyme find useful in a variety of biotechnological fields such as food and dairy (cheese ripening, flavour development, detergent, pharmaceutical (naproxen, ibuprofen, agrochemical (insecticide, pesticide and oleochemical (fat and oil hydrolysis, biosurfactant synthesis industries. Lipolytic enzyme can be further used in many newer areas where they can serve as potential biocatalysts.

  10. Extracellular α-Galactosidase from Trichoderma sp. (WF-3: Optimization of Enzyme Production and Biochemical Characterization

    Directory of Open Access Journals (Sweden)

    Aishwarya Singh Chauhan

    2015-01-01

    Full Text Available Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0 for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (Km, maximum rate of reaction (Vmax, Kcat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources.

  11. Belowground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soil.

    Science.gov (United States)

    Kaiser, Christina; Koranda, Marianne; Kitzler, Barbara; Fuchslueger, Lucia; Schnecker, Jörg; Schweiger, Peter; Rasche, Frank; Zechmeister-Boltenstern, Sophie; Sessitsch, Angela; Richter, Andreas

    2010-08-01

    *Plant seasonal cycles alter carbon (C) and nitrogen (N) availability for soil microbes, which may affect microbial community composition and thus feed back on microbial decomposition of soil organic material and plant N availability. The temporal dynamics of these plant-soil interactions are, however, unclear. *Here, we experimentally manipulated the C and N availability in a beech forest through N fertilization or tree girdling and conducted a detailed analysis of the seasonal pattern of microbial community composition and decomposition processes over 2 yr. *We found a strong relationship between microbial community composition and enzyme activities over the seasonal course. Phenoloxidase and peroxidase activities were highest during late summer, whereas cellulase and protease peaked in late autumn. Girdling, and thus loss of mycorrhiza, resulted in an increase in soil organic matter-degrading enzymes and a decrease in cellulase and protease activity. *Temporal changes in enzyme activities suggest a switch of the main substrate for decomposition between summer (soil organic matter) and autumn (plant litter). Our results indicate that ectomycorrhizal fungi are possibly involved in autumn cellulase and protease activity. Our study shows that, through belowground C allocation, trees significantly alter soil microbial communities, which may affect seasonal patterns of decomposition processes.

  12. Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate

    Directory of Open Access Journals (Sweden)

    SRI BUDIARTI

    2007-03-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC causes gastrointestinal infections in human. EPEC invasion was initiated by attachment and aggressive colonization on intestinal surface. Attachment of EPEC alter the intestine mucosal cells. Despite this, the pathogenic mechanism of EPEC infectior has not been fully understood. This research hypothesizes that extracellular proteolytic enzymes is necessary for EPEC colonization. The enzyme is secreted into gastrointestinal milieu and presumably destroy mucus layer cover the gastrointestinal tract. The objective of this study was to assay EPEC extracellular protease enzyme by using mucin substrate. The activity of EPEC extracellular proteolytic enzyme on 1% mucin substrate was investigated. Non-pathogenic E. coli was used as a negative control. Positive and tentative controls were Yersinia enterocolitica and Salmonella. Ten EPEC strains were assayed, seven of them were able to degrade mucin, and the highest activity was produced by K1.1 strain. Both positive and tentative controls also showed the ability to digest 0.20% mucin.

  13. Lignin-degrading enzyme activities.

    Science.gov (United States)

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

    2012-01-01

    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

  14. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  15. POTENTIAL USE OF AN EXTRACELLULAR ENZYME OF a-AMYLASE FROM INDIGENOUS INDONESIAN MESOPHILIC BACTERIA

    Directory of Open Access Journals (Sweden)

    Puji Lestari

    2013-04-01

    Full Text Available Amylase enzyme has a great significance for industrial usages in  Indonesia. However, this enzyme is still imported. The use of bacteria in biotechnological process of industrial products such as enzyme production has stimulated the exploration of extracellular amylase producing  bacteria. This study aimed to identify and analyze the potential use of amylolytic bacterial enzymes for hydrolyzing cassava starch. Two bacterial isolates, i.e. MII-10 and DKW-8 originated from Indonesia soil were identified based on their morphological, physiological and biochemical properties according to the standard protocol. The isolates were then  cultivated on fermentation medium and their growth pattern and  enzymatic assays were observed. The acetone-precipitated crude enzyme harvested based on predetermined cultivation time was used for  enzymatic hydrolysis product characterization on cassava starch using thin layer chromatography (TLC. The results showed that the mesophilicbacteria isolates (MII-10 and DKW-8 were belonged to Bacillus licheniformis. The maximum bacterial cell growth and enzyme activity were reached at 48 hours after incubation. The MII-10 isolate was found more stable than DKW-8 in producing amylase enzyme. Amylase produced by the MII-10 and DKW- 8 isolates was identified to be an endo-a-amylase as confirmed by oligosaccharides and dextrin of the random hydrolysisproducts. Relatively high dextrose equivalence (DE value of a-amylase of MII-10 (DE of 9.96 suggests that the enzyme is prospective for  saccharification of starchy material in glucose syrup industry.

  16. Potential of extracellular enzymes from Trametes versicolor F21a in Microcystis spp. degradation.

    Science.gov (United States)

    Du, Jingjing; Pu, Gaozhong; Shao, Chen; Cheng, Shujun; Cai, Ji; Zhou, Liang; Jia, Yong; Tian, Xingjun

    2015-03-01

    Studies have shown that microorganisms may be used to eliminate cyanobacteria in aquatic environments. The present study showed that the white-rot fungus Trametes versicolor F21a could degrade Microcystis aeruginosa. After T. versicolor F21a and Microcystis spp. were co-incubated for 60h, >96% of Microcystis spp. cells were degraded by T. versicolor F21a. The activities of extracellular enzymes showed that cellulase, β-glucosidase, protease, and laccase were vital to Microcystis spp. degradation in the early stage (0h to 24h), while β-glucosidase, protease, laccase, and manganese peroxidase in the late stage (24h to 60h). The positive and significant correlation of the degradation rate with these enzyme activities indicated that these enzymes were involved in the degradation rate of Microcystis spp. cells at different phases. It suggested that the extracellular enzymes released by T. versicolor F21a might be vital to Microcystis spp. degradation. The results of this study may be used to develop alternative microbial control agents for cyanobacterial control. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Extracellular Degradative Enzymes from Pleurotus pulmonarius Cultivated on Various Solid Cellulose- Radioactive Waste Simulates

    International Nuclear Information System (INIS)

    Abd El-Aziz, S.M.; El-Sayad, H.; Abu El- Soud, S.M.; Awad Alah, O.A.; Eskander, S.B.

    2008-01-01

    The present work was devoted to search the behavior of some extracellular enzymes secreted by P. pulmonarius during the bioremediation process of some cellulose based solid radioactive waste simulates. Four categories of this group, namely contaminated protective clothes, spent paper, and ruined cotton and mixture of them were subject to the fungal biodegradation and the variations in P. pulmonarius cellulase, xylanase and laccase enzymes activates were followed during three microbial growing stages. In addition, the changes in reducing sugars and total protein as end products of the degradation process were determined. Also the variations in both the secreted enzymes and the metabolism end products were measured as function of exposing the inoculated P. pulmonarius spawns to increasing doses of gamma irradiation(0.0,0.1,0.25,0.5,0.75,1.0,2.0 kGy). Based on the data so far obtained, it could be stated that the extracellular cellulase enzyme and total protein in the degraded substrate were increased throughout the whole incubation period for all types of cellulose based waste. In addition, it have been concluded that the enzymatic activities and consequently the biodegradation of the cellulose based solid radioactive simulates is enhanced by the gamma irradiation up to the dose 0.75 kGy

  18. 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 ...

  19. Screening for Extracellular Lipase Enzymes with Transesterification Capacity in Mucoromycotina Strains

    Directory of Open Access Journals (Sweden)

    Alexandra Kotogán

    2014-01-01

    Full Text Available In this study, 169 zygomycetes fungal strains including some cold-tolerant isolates were screened for their extracellular lipolytic activity towards tributyrin. Nineteen of them were outstanding in their enzyme production as they developed the largest lipolytic halo around the colonies in plate tests. Mortierella alpina, M. echinosphaera, Mucor corticolus, Rhizomucor miehei, Rhizopus oryzae, Rh. stolonifer, Umbelopsis autotrophica, U. isabellina, U. ramanniana var. angulispora and U. versiformis were selected for further studies to characterise their lipolytic enzyme production in detail. In these assays, effect of Tween 80 and palm, soybean, sunflower, olive, extra virgin olive, wheat germ, corn germ, sesame seed, pumpkin seed and cottonseed oils on the enzyme activities was investigated, and wheat bran-based submerged and solid-state fermentations were also tested. Tween 80 and olive oil proved to be efficient inductors for lipolytic enzyme production, which was also enhanced when wheat bran was used as support. Addition of mineral salts and olive oil to the solid fermentation medium resulted in at least 1.5-fold increment in the enzyme activities of the crude extracts. Organic synthesis was also assayed by the selected lipases, in which enzymes from the fungi R. miehei, Rh. stolonifer and M. echinosphaera gave the best yields during transesterification reactions between p-nitrophenyl palmitate and ethanol.

  20. Extracellular complex of chitinolytic enzymes of Clostridium paraputrificum strain J4 separated by membrane ultrafiltration.

    Science.gov (United States)

    Tishchenko, G; Koppová, I; Simůnek, J; Dohnálek, J

    2010-07-01

    Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing beta-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in%) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40-60 degrees C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.

  1. Photoperiodism and Enzyme Activity

    Science.gov (United States)

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  2. Large propeptides of fungal β-N-acetylhexosaminidases are novel enzyme regulators that must be intracellularly processed to control activity, dimerization, and secretion into the extracellular environment

    Czech Academy of Sciences Publication Activity Database

    Plíhal, Ondřej; Sklenář, Jan; Hofbauerová, Kateřina; Novák, Petr; Man, Petr; Pompach, Petr; Kavan, Daniel; Ryšlavá, H.; Weignerová, Lenka; Charvátová, Andrea; Křen, Vladimír; Bezouška, Karel

    2007-01-01

    Roč. 46, - (2007), s. 2719-2734 ISSN 0006-2960 R&D Projects: GA MŠk LC545; GA MŠk LC06010; GA ČR GA203/04/1045; GA ČR GA203/05/0172; GA ČR GA204/06/0771; GA AV ČR IAA5020403 Institutional research plan: CEZ:AV0Z50200510 Keywords : enzyme regulator * filamentous fungi * hex Subject RIV: EE - Microbiology, Virology Impact factor: 3.368, year: 2007

  3. A model of extracellular enzymes in free-living microbes: Which strategy pays off?

    DEFF Research Database (Denmark)

    Traving, Sachia J; Thygesen, Uffe Høgsbro; Riemann, Lasse

    2015-01-01

    An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes...... must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy...

  4. Extracellular enzyme production and phylogenetic distribution of yeasts in wastewater treatment systems.

    Science.gov (United States)

    Yang, Qingxiang; Zhang, Hao; Li, Xueling; Wang, Zhe; Xu, Ying; Ren, Siwei; Chen, Xuanyu; Xu, Yuanyuan; Hao, Hongxin; Wang, Hailei

    2013-02-01

    The abilities of yeasts to produce different extracellular enzymes and their distribution characteristics were studied in municipal, inosine fermentation, papermaking, antibiotic fermentation, and printing and dyeing wastewater treatment systems. The results indicated that of the 257 yeasts, 16, 14, 55, and 11 produced lipase, protease, manganese dependant peroxidase (MnP), and lignin peroxidase (LiP), respectively. They were distributed in 12 identified and four unidentified genera, in which Candida rugosa (AA-M17) and an unidentified Saccharomycetales (AA-Y5), Pseudozyma sp. (PH-M15), Candida sp. (MO-Y11), and Trichosporon montevideense (MO-M16) were shown to have the highest activity of lipase, protease, Mnp, and LiP, respectively. No yeast had amylase, cellulose, phytase, or laccase activity. Although only 60 isolates produced ligninolytic enzymes, 249 of the 257 yeasts could decolorize different dyes through the mechanism of biodegradation (222 isolates) or bio-sorption. The types of extracellular enzymes that the yeasts produced were significantly shaped by the types of wastewater treated. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Patterns of extracellular enzyme activities and microbial metabolism in an Arctic fjord of Svalbard and in the northern Gulf of Mexico: contrasts in carbon processing by pelagic microbial communities

    Directory of Open Access Journals (Sweden)

    Carol eArnosti

    2013-10-01

    Full Text Available The microbial community composition of polar and temperate ocean waters differs substantially, but the potential functional consequences of these differences are largely unexplored. We measured bacterial production, glucose metabolism, and the abilities of microbial communities to hydrolyze a range of polysaccharides in an Arctic fjord of Svalbard (Smeerenburgfjord, and thus to initiate remineralization of high-molecular weight organic matter. We compared these data with similar measurements previously carried out in the northern Gulf of Mexico in order to investigate whether differences in the spectrum of enzyme activities measurable in Arctic and temperate environments are reflected in ‘downstream’ aspects of microbial metabolism (metabolism of monomers and biomass production. Only 4 of 6 polysaccharide substrates were hydrolyzed in Smeerenburgfjord; all were hydrolyzed in the upper water column of the Gulf. These patterns are consistent on an interannual basis. Bacterial protein production was comparable at both locations, but the pathways of glucose utilization differed. Glucose incorporation rate constants were comparatively higher in Svalbard, but glucose respiration rate constants were higher in surface waters of the Gulf. As a result, at the time of sampling ca. 75% of the glucose was incorporated into biomass in Svalbard, but in the northern Gulf of Mexico most of the glucose was respired to CO2. A limited range of enzyme activities is therefore not a sign of a dormant community or one unable to further process substrates resulting from extracellular enzymatic hydrolysis. The ultimate fate of carbohydrates in marine waters, however, is strongly dependent upon the specific capabilities of heterotrophic microbial communities in these disparate environments.

  6. Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298

    Science.gov (United States)

    Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.

    2017-02-01

    Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

  7. Variability in the production of extracellular enzymes by entomopathogenic fungi grown on different substrates

    Directory of Open Access Journals (Sweden)

    Elio Gomes Fernandes

    2012-06-01

    Full Text Available Entomopathogenic fungi are important controllers of pest-insects populations in agricultural production systems and in natural environment. These fungi have enzymatic machinery which involve since the recognition and adherence of spores in their hosts culminating with infection and death of these insects. The main objective of this study was to analyzed extracellular enzyme production of the fungi strains Beauveria bassiana, Metarhizium anisopliae and Paecilomyces sp when cultured on substrates. These fungi were grown in minimal media containing specific substrates for the analysis of different enzymes such as amylases, cellulases, esterases, lipases, proteases (gelatin and caseinase, pectinases and cuticles of Musca domestica larvae and adults. All the assays were performed with and without the presence of dextrose in the culture media. The quantification of enzyme activity was performed by the ratio of halo / colony (H/C and the results subjected to variance analysis level of 5% (ANOVA followed by post-Tukey test. All strains were positive for lipase and also they showed a high significant enzyme production for gelatin at concentrations of 4 and 1%. B. bassiana and Paecilomyces sp. were positive for amylase, pectinase and caseinase, and only Paecilomyces sp. showed cellulase activity.

  8. Effect of passive transport of water through plasma membrane in production of extracellular enzyme.

    Science.gov (United States)

    Mahmoodi, M; Najafpour, G D; Mohammadi, M

    2017-02-01

    In this article, availability and control of water in solid-state fermentation (SSF) were investigated. Based on passive transport of water through plasma membranes, a new model was proposed for calculation and control of water activities in the mixture of solids. The validity of theoretical model and accuracy of the proposed model were proved by experimental data. This model was used for production of pectinases via mixed-SSF with the aid of a rotary drum bioreactor. It was found that in case of extracellular enzyme production, the new model is in good agreement with experimental data for the control of water activities in the mixed-SSF. Exact control of water activity in SFF, the production of endo- and exo-pectinases was relatively enhanced. Based on theoretical view point, the prominence of this new model in control of water activity was also proved.

  9. Seasonality of fibrolytic enzyme activity in herbivore microbial ...

    African Journals Online (AJOL)

    Fibre (cellulose, hemicellulose and lignin) is the most abundant polysaccharide in nature and is hydrolysed by gut micro-organisms of herbivores because they can produce a set of extracellular enzymes. This study examined seasonal changes in the fibrolytic enzyme activity of microbial ecosystems of five herbivores ...

  10. Managing Brain Extracellular K(+) during Neuronal Activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    isoform compositions of the Na(+)/K(+)-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K(+) from neurons, whereas the neurons themselves become the primary K......During neuronal activity in the brain, extracellular K(+) rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K(+) is the Na(+)/K(+)-ATPase, although the relative involvement and physiological impact of the different subunit......(+) absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na(+)/K(+)-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic...

  11. A model of extracellular enzymes in free-living microbes: which strategy pays off?

    Science.gov (United States)

    Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

    2015-11-01

    An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

    Directory of Open Access Journals (Sweden)

    Shuping Qin

    Full Text Available Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v and power density  =  15 watt ml(-1], the activity of alkaline phosphomonoesterase (phosphatase in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80% of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

  13. Enzyme activity and expression pattern of intra- and extracellular chitinase and β-1,3-glucanase of Wickerhamomyces anomalus EG2 using glycol chitin and glucan-containing high polymer complex.

    Science.gov (United States)

    Hong, Sin-Hyoung; Song, Yong-Su; Seo, Dong-Jun; Kim, Kil-Yong; Jung, Woo-Jin

    2017-12-01

    We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

    Science.gov (United States)

    Liu, Lin; Gong, Weili; Sun, Xiaomeng; Chen, Guanjun; Wang, Lushan

    2018-02-07

    Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of β-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

  15. [Isolation of protoplasts from vegetable tissues using extracellular lytic enzymes from fusarium oxysporum f.sp. melonis].

    Science.gov (United States)

    Alconada, T M; Martínez, M J

    1995-01-01

    Fusarium oxysporum f.sp. melonis, a pathogen of melon (Cucumis melo L.), was grown in shaken cultures at 26 degrees C in a mineral salts medium containing glucose, xylan and apple pectin as carbon sources. The extracellular enzymic complex obtained from these cultures showed lytic activity on plant tissues, causing maceration of melon fruits, potato tubers and carrot roots. Protoplasts were isolated from melon fruits when the maceration was carried out under appropriate osmotic conditions. This fact suggest a possible relationship between the enzymes produced by Fusarium oxysporum f.sp. melonis and their pathogenicity on melon plants.

  16. Bioremediation of Direct Blue 14 and Extracellular Ligninolytic Enzyme Production by White Rot Fungi: Pleurotus Spp.

    Science.gov (United States)

    Singh, M. P.; Vishwakarma, S. K.; Srivastava, A. K.

    2013-01-01

    In the present investigation, four species of white rot fungi (Pleurotus), that is, P. flabellatus, P. florida, P. ostreatus and P. sajor-caju were used for decolorization of direct blue 14 (DB14). Among all four species of Pleurotus, P. flabellatus showed the fastest decolorization in petri plates on different concentration, that is, 200 mg/L, 400 mg/L, and 600 mg/L. All these four species were also evaluated for extracellular ligninolytic enzymes (laccase and manganese peroxidase) production and it was observed that the twelve days old culture of P. flabellatus showed the maximum enzymatic activity, that is, 915.7 U/mL and 769.2 U/mL of laccase and manganese peroxidase, respectively. Other three Pleurotus species took more time for dye decolorization and exhibited less enzymatic activities. The rate of decolorization of DB14 dye solution (20 mg/L) by crude enzymes isolated from P. flabellatus was very fast, and it was observed that up to 90.39% dye solution was decolorized in 6 hrs of incubation. PMID:23841054

  17. THE MOBILIZATION AND EXTRACELLULAR RELEASE OF GRANULAR ENZYMES FROM HUMAN LEUKOCYTES DURING PHAGOCYTOSIS

    Science.gov (United States)

    Wright, Daniel G.; Malawista, Stephen E.

    1972-01-01

    The importance of granular (lysosomal) enzymes from neutrophils in producing the tissue damage of acute inflammation has been suggested by much indirect and some direct evidence. This study has investigated the kinetics of release and subsequent fate of granular enzymes from phagocytizing human leukocytes The following observations are made: (a) During phagocytosis, the granular enzyme lysozyme is released from leukocytes into the extracellular medium. (b) Release of lysozyme increases as phagocytic challenge increases, but attains a maximum. (c) Release of lysozyme accompanies phagocytosis and is not a delayed event. (d) The lack of release of a nongranular enzyme, lactic dehydrogenase, indicates that cell damage is not a necessary condition of enzyme release. (e) Like lysozyme, β-glucuronidase is released from phagocytizing leukocytes. Acid α-naphthyl phosphatase and cathepsin also appear to be released, but are not found in appreciable amounts in the extracellular medium, in part because of their lability in solution. These results support the concept that extracellular release of granular enzymes may be a useful secretory function of inflammatory leukocytes which becomes damaging to the host in certain circumstances. PMID:5028260

  18. Small-scale distribution of extracellular enzymes, fungal, and bacterial biomass in Quercus petraea forest topsoil

    Czech Academy of Sciences Publication Activity Database

    Baldrian, Petr; Merhautová, Věra; Cajthaml, Tomáš; Petránková, Mirka; Šnajdr, Jaroslav

    2010-01-01

    Roč. 46, č. 7 (2010), s. 717-726 ISSN 0178-2762 R&D Projects: GA MŠk LC06066; GA MŠk(CZ) ME10152; GA MZe QH72216 Institutional research plan: CEZ:AV0Z50200510 Keywords : Decomposition * Extracellular enzymes * Forest soil Subject RIV: EE - Microbiology, Virology Impact factor: 2.156, year: 2010

  19. Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

    Science.gov (United States)

    Gómez-Arreaza, Amaranta; Acosta, Hector; Quiñones, Wilfredo; Concepción, Juan Luis; Michels, Paul A M; Avilán, Luisana

    2014-02-01

    In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. [Extracellular hydrolytic enzymes produced by entomopathogenic fungi--role in an infection process].

    Science.gov (United States)

    Włóka, Emilia

    2011-01-01

    Entomopathogenic fungi have a great potential in biological control of insect pest population. Fungal pathogens are promising source of insecticides and notable alterative to chemical pesticides. These fungi possess a unique mechanism of insects paralysis. As natural enemies of insects they attack direct host cuticle via a combination of mechanical pressure and cuticle-degrading enzymes. Entomopathogenic fungi produce several proteo-, chitino- and lipolytic enzymes, which are accepted as key factors in insect mycosis. The role of extracellular enzymes in pathogenesis is still not well understood. Profound understanding the mechanisms of insect paralysis by entomopathogenic fungi will help in the production of safer for environment and more efficiency mycoinsecticides.

  1. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression. Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells. © 2017 American Heart Association, Inc.

  2. Effects of dilution rate on biomass and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture.

    Science.gov (United States)

    Greenman, J; Holland, K T

    1985-07-01

    Propionibacterium acnes, P. avidum and P. granulosum were grown in continuous culture at a range of dilution rates on a semi-synthetic medium. Dilution rates were chosen to allow the bacteria to grow at the same relative growth rates as compared to their respective mumax values. The steady-state levels and production rates of biomass and extracellular enzymes were determined. The lipase and hyaluronate lyase of P. granulosum and the proteolytic activity of P. acnes and P. avidum were growth linked enzymes (i.e. they were produced at constant amounts per unit of biomass). In contrast, the lipase, hyaluronate lyase and acid phosphatase of P. acnes and the lipase of P. avidum were shown to be non-growth linked enzymes.

  3. Temperature sensitivity of extracellular enzyme kinetics in subtropical wetland soils under different nutrient and water level conditions

    Science.gov (United States)

    Goswami, S.; Inglett, K.; Inglett, P.

    2012-12-01

    Microbial extracellular enzymes play an important role in the initial steps of soil organic matter decomposition and are involved in regulating nutrient cycle processes. Moreover, with the recent concern of climate change, microbial extracellular enzymes may affect the functioning (C losses, C sequestration, greenhouse gas emissions, vegetation changes) of different ecosystems. Hence, it is imperative to understand the biogeochemical processes that may be climate change sensitive. Here, we have measured the Michaelis Menten Kinetics [maximal rate of velocity (Vmax) and half-saturation constant (Km)] of 6 enzymes involved in soil organic matter decomposition (phosphatase, phosphodiesterase, β-D-glucosidase, cellobiohydrolase, leucine aminopeptidase, N-Acetyl-β-D glucosaminidase) in different nutrient(P) concentration both aerobically and anaerobically in Everglade water conservation area 2A (F1, F4-slough and U3-slough). Temperature sensitivity of different enzymes is assessed within soil of different P concentrations. We hypothesized that the temperature sensitivity of the enzyme changes with the biogeochemical conditions including water level and nutrient condition. Furthermore, we have tested specific hypothesis that higher P concentration will initiate more C demand for microbes leading to higher Vmax value for carbon processing enzymes in high P site. We found temperature sensitivity of all enzymes for Vmax and Km under both aerobic and anaerobic condition ranges from 0.6 to 3.2 for Vmax and 0.5 to 2.5 for Km. Q10 values of Km for glucosidase indicate more temperature sensitivity under anaerobic condition. Under aerobic condition higher temperature showed significant effect on Vmax for bisphosphatase between high P and low P site. Decreasing P concentration from F1 site to U3-S site had showed significant effect in all temperature on carbon processing enzyme. This suggests that in high P site, microbes will use more carbon-processing enzyme to get more carbon

  4. EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

    DEFF Research Database (Denmark)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G

    2010-01-01

    an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across...... the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens...... are targeted primarily towards partial degradation of plant cell walls, reflecting a plesiomorphic state of non-domesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major...

  5. Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain.

    Science.gov (United States)

    Cherif, Slim; Mnif, Sami; Hadrich, Fatma; Abdelkafi, Slim; Sayadi, Sami

    2011-11-11

    Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C). The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry), was used as the sole carbon source. A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment.

  6. Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain

    Directory of Open Access Journals (Sweden)

    Cherif Slim

    2011-11-01

    Full Text Available Abstract Background Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C. The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry, was used as the sole carbon source. Conclusion A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment

  7. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  8. NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation.

    Science.gov (United States)

    Sinsuwan, S; Rodtong, S; Yongsawatdigul, J

    2007-06-01

    Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.

  9. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  10. Quorum sensing in marine snow and its possible influence on production of extracellular hydrolytic enzymes in marine snow bacterium Pantoea ananatis B9.

    Science.gov (United States)

    Jatt, Abdul Nabi; Tang, Kaihao; Liu, Jiwen; Zhang, Zenghu; Zhang, Xiao-Hua

    2015-02-01

    Marine snow is a continuous shower of organic and inorganic detritus, and plays a crucial role in transporting materials from the sea surface to the deep ocean. The aims of the current study were to identify N-acyl homoserine lactone (AHL)-based quorum sensing (QS) signaling molecules directly from marine snow particles and to investigate the possible regulatory link between QS signals and extracellular hydrolytic enzymes produced by marine snow bacteria. The marine snow samples were collected from the surface water of China marginal seas. Two AHLs, i.e. 3OC6-HSL and C8-HSL, were identified directly from marine snow particles, while six different AHL signals, i.e. C4-HSL, 3OC6-HSL, C6-HSL, C10-HSL, C12-HSL and C14-HSL were produced by Pantoea ananatis B9 inhabiting natural marine snow particles. Of the extracellular hydrolytic enzymes produced by P. ananatis B9, alkaline phosphatase activity was highly enhanced in growth medium supplemented with exogenous AHL (C10-HSL), while quorum quenching enzyme (AiiA) drastically reduced the enzyme activity. To our knowledge, this is the first report revealing six different AHL signals produced by P. ananatis B9 and AHL-based QS system enhanced the extracellular hydrolytic enzyme in P. ananatis B9. Furthermore, this study first time revealing 3OC6-HSL production by Paracoccus carotinifaciens affiliated with Alphaproteobacteria. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase and molecular sequencing of selected strains

    Directory of Open Access Journals (Sweden)

    Mauro Cintra Giudice

    2012-09-01

    Full Text Available A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum. The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

  12. Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1989-01-01

    of an enzyme, and it is mediated by the Na+ -K+ pump located in the plasma membrane. It is demonstrated that the activity of the Na+ -K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1-1.2 mmol l-1). The possibility is discussed that calcium-deprivation may increase the pump...... activity by increasing the permeability of the plasma membrane for Na+....

  13. Useful Extracellular Activity of Bacteria Isolated from Bhitarkanika Mangrove Ecosystem of Orissa Coast

    Directory of Open Access Journals (Sweden)

    Gupta, N.

    2007-01-01

    Full Text Available This paper describes the isolation of bacteria from Bhitarkanika mangrove ecosystem of Orissa and screening for their antifungal properties against five pathogenic fungi, extracellular enzyme activity like amylase, protease and lipase, and phosphate solubilization capacity. From 567 bacterial isolates obtained, 26 bacterial isolates have exhibited wide spectrum antifungal activity against all five fungi tested. Most of the bacterial isolates were found to be amylase and protease producers. In present study, 41 lipase producers and 33 phosphate solubilisers were also found. Bacterial isolates from plant origin exhibited all the four extracellular enzyme activity except lipase. The maximum % occurrence of phosphate solubilisers was found in soil and plant system of mangrove. This screening study opens an avenue to work with some of the potent strains for useful product formation at large scale.

  14. Extracellular proteolytic activity of Deinococcus geothermalis ...

    African Journals Online (AJOL)

    Nonionic detergents like Triton X-100 and Tween 80 did not affect catalytic properties. It suggested that the enzyme produced by D. geothermalis could be used as a component of detergents. Keywords: Deinococcus geothermalis, alkaline protease, detergents, thermostability. African Journal of Biotechnology Vol. 12(25) ...

  15. Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid.

    Science.gov (United States)

    Khan, Abdul Latif; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Al-Farsi, Zainab; Al-Mamari, Aza; Waqas, Muhammad; Asaf, Sajjad; Elyassi, Ali; Mabood, Fazal; Shin, Jae-Ho; Lee, In-Jung

    2016-01-01

    Boswellia sacra, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%), Chaetomiaceae (17.6%), Incertae sadis (29.5%), Aureobasidiaceae (17.6%), Nectriaceae (5.9%) and Sporomiaceae (17.6%) from the phylloplane (leaf) and caulosphere (stem) of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33%) than the stem (0.262%). The Shannon-Weiner diversity index was H' 0.8729, while Simpson index was higher in the leaf (0.583) than in the stem (0.416). Regarding the endophytic fungi's potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, Penicillum citrinum BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 μM-1min-1mL) and cellulases (62.11±1.6 μM-1min-1mL), whereas Preussia sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 μM-1min-1mL) and phosphatases (3.46±0.31μM-1min-1mL) compared to other strains. Aureobasidium sp. BSS6 and Preussia sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways). Preussia sp. BSL10 was applied to the host B. sacra tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin could

  16. Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid.

    Directory of Open Access Journals (Sweden)

    Abdul Latif Khan

    Full Text Available Boswellia sacra, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%, Chaetomiaceae (17.6%, Incertae sadis (29.5%, Aureobasidiaceae (17.6%, Nectriaceae (5.9% and Sporomiaceae (17.6% from the phylloplane (leaf and caulosphere (stem of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33% than the stem (0.262%. The Shannon-Weiner diversity index was H' 0.8729, while Simpson index was higher in the leaf (0.583 than in the stem (0.416. Regarding the endophytic fungi's potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, Penicillum citrinum BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 μM-1min-1mL and cellulases (62.11±1.6 μM-1min-1mL, whereas Preussia sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 μM-1min-1mL and phosphatases (3.46±0.31μM-1min-1mL compared to other strains. Aureobasidium sp. BSS6 and Preussia sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways. Preussia sp. BSL10 was applied to the host B. sacra tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin

  17. Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration

    Directory of Open Access Journals (Sweden)

    García-Carmona Francisco F

    2002-03-01

    Full Text Available Abstract Background The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114. Results A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation, 36% of which was extracellular (65% after activation. The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration. Conclusions The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

  18. Subterranean Microhabitat Dependent Intra Versus Extracellular Enzyme Secretion Capabilities of Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Jayant Biswas

    2015-04-01

    Full Text Available Deinococcus radiodurans is one of the most yet discovered extremophilic microbe, the isolation of which from the various habitats of Kotumsar cave is always a matter of enticement to discover its ecological economics. In the present work we studied the intra versus extracellular alkaline protease and glucose isomerase secretion capabilities of Deinococcus radiodurans; KCB21, KCB50, KCB93 isolated from three distinct subterranean niches of Kotumsar cave. The selected niches/zones were the entrance zone, transient zone and the deep inner zone from where the soil sediments were collected to isolate the bacterial strains. The results revealed high extracellular alkaline protease activity from the Deinococcus radiodurans strain which was isolated from the deeper zones of the cave, whereas no such phenomenon was revealed for glucose isomerase. The possible reason for the obtained results has been discussed.

  19. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  20. Detoxification enzymes activities in deltamethrin and bendiocarb ...

    African Journals Online (AJOL)

    Detoxification enzymes activities in deltamethrin and bendiocarb resistant and susceptible malarial vectors ( Anopheles gambiae ) breeding in Bichi agricultural and residential sites, Kano state, Nigeria.

  1. Depth-Resolved Variations of Cultivable Bacteria and Their Extracellular Enzymes in the Water Column of the New Britain Trench

    Directory of Open Access Journals (Sweden)

    Qianfeng Liu

    2018-02-01

    Full Text Available Marine microorganisms and their extracellular enzymes (ECEs play an important role in the remineralization of organic material by hydrolyzing high-molecular-weight substrates to sizes sufficiently small to be transported through cell membrane, yet the diversity of the enzyme-producing bacteria and the types of ECEs involved in the degradation process are largely unknown. In this work, we investigated the diversity of cultivable bacteria and their ECEs and the potential activities of aminopeptidase in the water column at eight different depths of the New Britain Trench. There was a great diversity of cultivable bacteria and ECEs, and depth appears an important driver of the diversity. The 16S rRNA sequence analysis revealed that the cultivable bacteria were affiliated mostly with the phyla Proteobacteria and Actinobacteria, and the predominant genera were Pseudoalteromonas (62.7% and Halomonas (17.3%. Moreover, 70.7% of the isolates were found to produce hydrolytic zone on casein and gelatin plates, in which Pseudoalteromonas was the predominant group, exhibiting relatively high protease production. Inhibitor analysis showed that the extracellular proteases from the isolated bacteria were serine proteases in the surface water and metalloproteases in the deep water. Meanwhile, the Vmax and Km of aminopeptidase exhibited a maximum in the surface water and low values in the deep bathy- and abyssopelagic water, indicating lower rates of hydrolysis and higher substrate affinity in the deeper waters. These results shed new insights into the diversity of the cultivable bacteria and bacterial ECEs and their likely biogeochemical functions in the trench environment.

  2. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    Science.gov (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  3. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  4. Enzyme inhibition activities of Andrachne cardifolia Muell.

    Science.gov (United States)

    Ahmad, Bashir; Shah, S M Hassan; Bashir, Shumaila; Shah, Jehandar

    2007-04-01

    The crude methanolic extract and various fractions of Andrachne cardifolia Muell, including chloroform, ethyl acetate and n-butanol fractions were subjected to in vitro enzyme inhibition activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (40-89%) was shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (40-71%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated poor to significant activity (25-73%) against acetylcholinesterase and no activity against urease.

  5. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

    Directory of Open Access Journals (Sweden)

    Daryl L Moorhead

    2013-08-01

    Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

  6. Characterization of the protease activity that cleaves the extracellular domain of β-dystroglycan

    International Nuclear Information System (INIS)

    Zhong Di; Saito, Fumiaki; Saito, Yuko; Nakamura, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2006-01-01

    Dystroglycan (DG) complex, composed of αDG and βDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of βDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of βDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of βDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of βDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process

  7. Extracellular electrical activity from the photoreceptors of midge

    Indian Academy of Sciences (India)

    The ontogeny of photosensitivity has been studied in a holometabolous insect, the midge Chironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in ...

  8. Temperature and UV light affect the activity of marine cell-free enzymes

    Directory of Open Access Journals (Sweden)

    B. Thomson

    2017-09-01

    Full Text Available Microbial extracellular enzymatic activity (EEA is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells. Experiments were run to assess how cell-free enzymes (excluding microbes respond to ultraviolet radiation (UVR and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase, β-glucosidase, (BGase, and leucine aminopeptidase (LAPase. Environmentally relevant UVR (i.e. in situ UVR levels measured at our site reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C, likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

  9. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  10. Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

    Directory of Open Access Journals (Sweden)

    Boriana K Tchernookova

    Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

  11. Antimutagenic activity of oxidase enzymes

    International Nuclear Information System (INIS)

    Agabeili, R.A.

    1986-01-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity

  12. Effects of pH on biomass, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture.

    Science.gov (United States)

    Greenman, J; Holland, K T; Cunliffe, W J

    1983-05-01

    Three cutaneous propionibacteria, Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum, were grown in chemostats using semi-synthetic medium at various pH values. Growth occurred between pH 4.5 and 7.5 for P. acnes and pH 5.0 and 8.0 for P. avidum and P. granulosum. The highest mumax was at pH 6.0 for the three species. Maximum biomass production was obtained at pH 6.0 for P. acnes and P. avidum and at pH 7.5 for P. granulosum. Extracellular enzyme production occurred over the entire pH growth range when denaturation of the enzymes was taken into account. However, detectable activity of the enzymes was found in a narrower range of pH due to the denaturation of the enzymes at low or high pH values. The highest production of enzymes occurred at pH values between 5.0 and 6.0, apart from the production of hyaluronate lyase of P. granulosum (pH 6.0 to 7.0) and the proteinase of P. acnes and P. avidum (pH 5.0 to 7.5). Propionibacterium acnes produced a lipase, hyaluronate lyase, phosphatase and proteinase activity. Propionibacterium avidum produced a lipase and proteinase activity. Propionibacterium granulosum produced a lipase and hyaluronate lyase.

  13. Enzyme Enzyme activities in relation to sugar accumulation in tomato

    International Nuclear Information System (INIS)

    Alam, M.J.; Rahman, M.H.; Mamun, M.A.; Islam, K.

    2006-01-01

    Enzyme activities in tomato juice of five different varieties viz. Ratan, Marglove, BARI-1, BARI-5 and BARI-6, in relation to sugar accumulation were investigated at different maturity stages. The highest amount of invertase and beta-galactosidase was found in Marglove and the lowest in BARI- 6 at all maturity stages. Total soluble sugar and sucrose contents were highest in BARI-1 and lowest in BARI-6. The activity of amylase was maximum in Ratan and minimum in Marglove. Protease activity was highest in Ratan and lowest in BARI-6. BARI-1 contained the highest cellulase activity and the lowest in BARI-5. The amount of total soluble sugar and sucrose increased moderately from premature to ripe stage. The activities of amylase and cellulase increased up to the mature stage and then decreased drastically in the ripe stage. The activities of invertase and protease increased sharply from the premature to the ripe stage while the beta-galactosidase activity decreased remarkably. No detectable amount of reducing sugar was present in the premature stage in all cultivars of tomato but increased thereafter upto the ripe stage. The highest reducing sugar was present in BARI-5 in all of the maturity stages. (author)

  14. Problems with extracellular recording of electrical activity in gastrointestinal muscle.

    Science.gov (United States)

    Sanders, Kenton M; Ward, Sean M; Hennig, Grant W

    2016-12-01

    Motility patterns of the gastrointestinal tract are important for efficient processing of nutrients and waste. Peristalsis and segmentation are based on rhythmic electrical slow waves that generate the phasic contractions fundamental to gastrointestinal motility. Slow waves are generated and propagated actively by interstitial cells of Cajal (ICC), and these events conduct to smooth muscle cells to elicit excitation-contraction coupling. Extracellular electrical recording has been utilized to characterize slow-wave generation and propagation and abnormalities that might be responsible for gastrointestinal motility disorders. Electrode array recording and digital processing are being used to generate data for models of electrical propagation in normal and pathophysiological conditions. Here, we discuss techniques of extracellular recording as applied to gastrointestinal organs and how mechanical artefacts might contaminate these recordings and confound their interpretation. Without rigorous controls for movement, current interpretations of extracellular recordings might ascribe inaccurate behaviours and electrical anomalies to ICC networks and gastrointestinal muscles, bringing into question the findings and validity of models of gastrointestinal electrophysiology developed from these recordings.

  15. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  16. Antibacterial activity of dentine and pulp extracellular matrix extracts.

    Science.gov (United States)

    Smith, J G; Smith, A J; Shelton, R M; Cooper, P R

    2012-08-01

    To determine whether extracellular matrix (ECM) preparations from pulp (pECM) and dentine (dECM) possess antimicrobial activity. Dentine and pulp ECM preparations were isolated with 10% ethylenediaminetetraacetic acid (EDTA), pH 7.2 and sequential use of 0.5mol L(-1) NaCl, pH 11.7 and 0.1mol L(-1) tartaric acid, pH 2.0, respectively, with protease inhibitor inclusion throughout. Antimicrobial activity against Streptococcus mutans, Streptococcus oralis and Enterococcus faecalis was assessed using turbidity as a measure of bacteria growth. The cytotoxicity of the extracts on primary pulp cells was also determined by lactate dehydrogenase (LDH) release. Statistical analysis of data was performed using paired student's t-tests. Extracellular matrix extracts from the pulp and dentine showed antibacterial activity against three types of anaerobic bacteria associated with dental disease (Ppulpal cells at the concentrations used for antibacterial activity. The bacteriostatic antibacterial activity of pECM and dECM indicates that the release of these matrix molecules from pulp and dentine may contribute to defence responses during dental disease, treatment and repair. © 2012 International Endodontic Journal.

  17. Complete genome sequence of Bacillus sp. 275, producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes.

    Science.gov (United States)

    Gong, Gyeongtaek; Kim, Seil; Lee, Sun-Mi; Woo, Han Min; Park, Tai Hyun; Um, Youngsoon

    2017-07-20

    Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G+C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

    NARCIS (Netherlands)

    Braun, P; Bitter, W; Tommassen, J

    2000-01-01

    The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes

  19. Enzyme Activities in Waste Water and Activated Sludge

    DEFF Research Database (Denmark)

    Nybroe, Ole; Jørgensen, Per Elberg; Henze, Mogens

    1992-01-01

    measured as colony forming units of heterotrophic bacteria. A panel of four enzyme activity assays, α-glucosidase, alanine-aminopeptidase, esterase and dehydrogenase were used to characterize activated sludge and anaerobic hydrolysis sludge from a pilot scale plant. The enzymatic activity profiles were...... starch was, for instance, reflected in a high α-glucosidase activity. However, no obvious correlations between specific process parameters and enzyme activities were found....

  20. Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

    Science.gov (United States)

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

  1. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused CBHI.1 protein. Plate enzyme ...

  2. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused. CBHI.1 protein. Plate enzyme ...

  3. Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains

    Directory of Open Access Journals (Sweden)

    Lauren Mullen

    2018-03-01

    Full Text Available Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States and near Knoxville (TN, United States, between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase, which cleave N-terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases.

  4. A New Enzyme-linked Sorbent Assay (ELSA) to Quantify Syncytiotrophoblast Extracellular Vesicles in Biological Fluids

    NARCIS (Netherlands)

    Goehner, Claudia; Weber, Maja; Tannetta, Dionne S.; Groten, Tanja; Ploesch, Torsten; Faas, Marijke M.; Scherjon, Sicco A.; Schleussner, Ekkehard; Markert, Udo R.; Fitzgerald, Justine S.

    ProblemThe pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are

  5. Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

    Science.gov (United States)

    Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan

    2016-11-21

    During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating

  6. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  7. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  8. A novel extracellular multicopper oxidase from Phanerochaete chrysosporium with ferroxidase activity

    Science.gov (United States)

    Luis F. Larrondo; Loreto Salas; Francisco Melo; Rafael Vicuna; Daniel Cullen

    2003-01-01

    Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified...

  9. Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A activity in human lung fibroblasts

    Directory of Open Access Journals (Sweden)

    Cappello Francesco

    2007-03-01

    Full Text Available Abstract Background Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD. Methods In this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line exposed to cigarette smoke extract (CSE. Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms, and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting. Results CSE exposure at the doses used (1–10% did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h, while correlation analysis showed that this result was

  10. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  11. Enzyme Activities in Waste Water and Activated Sludge

    DEFF Research Database (Denmark)

    Nybroe, Ole; Jørgensen, Per Elberg; Henze, Mogens

    1992-01-01

    The purpose of the present study was to evaluate the potential of selected enzyme activity assays to determine microbial abundance and heterotrophic activity in waste water and activated sludge. In waste water, esterase and dehydrogenase activities were found to correlate with microbial abundance...... measured as colony forming units of heterotrophic bacteria. A panel of four enzyme activity assays, α-glucosidase, alanine-aminopeptidase, esterase and dehydrogenase were used to characterize activated sludge and anaerobic hydrolysis sludge from a pilot scale plant. The enzymatic activity profiles were...... distinctly different, suggesting that microbial populations were different, or had different physiological properties, in the two types of sludge. Enzyme activity profiles in activated sludge from four full-scale plants seemed to be highly influenced by the composition of the inlet. Addition of hydrolysed...

  12. Procedures and means for determining enzyme activities

    International Nuclear Information System (INIS)

    Hunger, H.; Behrendt, G.; Schmidt, G.

    1988-01-01

    Aim of the invention is an improved procedure for the determination of phosphorylating enzyme activity by using an improved means. Cellular extracts and cell lysates, resp., are contacted with surface carriers from natural and/or synthesized polymers, containing deprotonizable groups, in presence of 32 P-γ-ATP and, if necessary, of ATP. The phosphorylated antibiotic is evidenced by autoradiography or any other identifying procedure. Fields of application are molecular biology, genetic engineering, biotechnology and medical special fields

  13. Active endocannabinoids are secreted on extracellular membrane vesicles.

    Science.gov (United States)

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. © 2015 The Authors.

  14. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    Directory of Open Access Journals (Sweden)

    Aline B. M Vaz

    2011-09-01

    Full Text Available The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia, Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

  15. Distribution of extracellular enzymes in soils: Spatial heterogeneity and determining factors at various scales

    Czech Academy of Sciences Publication Activity Database

    Baldrian, Petr

    2014-01-01

    Roč. 78, č. 1 (2014), s. 11-18 ISSN 0361-5995 R&D Projects: GA ČR GAP504/12/1288; GA ČR GA13-06763S Institutional support: RVO:61388971 Keywords : enzyme * soil microflora * distribution Subject RIV: EE - Microbiology, Virology Impact factor: 1.721, year: 2014

  16. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

    Directory of Open Access Journals (Sweden)

    Joanna Homa

    Full Text Available Formation of extracellular traps (ETs capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA, histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i facilitating decondensation of chromatin by citrullination of histones, and (ii serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27 and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

  17. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  18. Extracellular complex of chitinolytic enzymes of Clostridium paraputrificum strain J4 separated by membrane ultrafiltration

    Czech Academy of Sciences Publication Activity Database

    Tishchenko, Galina; Koppová, Ingrid; Šimůnek, Jiří; Dohnálek, Jan

    2010-01-01

    Roč. 55, č. 4 (2010), s. 386-389 ISSN 0015-5632 R&D Projects: GA ČR(CZ) GA525/08/0803; GA ČR GA310/09/1407 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515 Keywords : chitinolytic enzymes * Clostridium paraputrificum * membrane diafiltration Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 0.977, year: 2010

  19. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

    Science.gov (United States)

    McKee, Marc D; Hoac, Betty; Addison, William N; Barros, Nilana M T; Millán, José L; Chaussain, Catherine

    2013-10-01

    As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  1. Purification and Characterization of Extracellular enzyme from Aspergillus fumigatus and Its Application on a pennisetum sp for enhanced glucose production

    Directory of Open Access Journals (Sweden)

    Sonali Mohapatra

    2017-12-01

    Full Text Available Aspergillus species are saprophytic fungi widely distributed in nature and are associated with a number of human diseases. The present study was investigated for production of extracellular cellulase from Aspergillus fumigatus which could be potentially used for degradation of cellulose in lignocellulosic biomass for bioethanol production. In the present work, A. fumigatus were grown in fungal basal medium and preserved at 30 °C for 72 h. The cellulase enzyme was filtered (using Whatman filter paper, precipitated (using ammonium sulphate, dialysed and then purified on a Sepharose 6B ion exchange column. The cellulase enzyme showed a purification of 0.4 fold and the molecular weight was determined as 100 kDa by SDS-PAGE. The optimum pH, temperature, incubation time of the enzyme was determined to be pH 7.0, 35 °C and 24 h respectively. The presence of metal ion Mn2+, followed by Ca2+ and Co2+ was found to increase the cellulase activity. Notably, the cellulase activity was not significantly affected in the presence of additives like EDTA, and Triton X-100 and β-mercaptoethanol. Response surface methodology was used to design optimisation experiments for saccharification of lignocellulosic biomass (hybrid napier grass and the response i.e. glucose yield was considered as the product. The glucose yield was considerably increased from 101.4 mg/g to 856.5 mg/g in the optimised conditions of 35°C, pH 5.2 with substrate concentration (ultrasono assisted alkali pretreated biomass of 3.5 g, with enzyme concentration of 3 ml was incubated for 24 h. Further, the statistical analysis using ANNOVA demonstrated a p- value of less than 0.005 and the R2 value of 90.18.

  2. Human mammospheres secrete hormone-regulated active extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Esperanza Gonzalez

    Full Text Available Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.

  3. Detection of enzyme activity in decontaminated spices of industrial use

    International Nuclear Information System (INIS)

    Müller, R.; Theobald, R.

    1995-01-01

    A range of decontaminated spices of industrial use have been examinated for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material [de

  4. 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.

  5. Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

    Science.gov (United States)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify

  6. The Effect of Catchment Urbanization on Nutrient Uptake and Biofilm Enzyme Activity in Lake Superior (USA) Tributary Streams

    Science.gov (United States)

    We used landscape, habitat, and chemistry variables, along with nutrient spiraling metrics and biofilm extracellular enzyme activity (EEA), to assess the response of streams to the level of urbanization within their catchments. For this study nine streams of similar catchment are...

  7. Larvicidal activity of extracellular secondary metabolites from a ...

    African Journals Online (AJOL)

    The main objective of this investigation was to find mosquito larvicidal secondary metabolites from a basidiomycete – Stereum species (JO5289) – against Aedes aegypti. The Stereum species (JO5289) was collected in July 2005 from undisturbed habitat in Londiani forest in Rift Valley province, Kenya. Extracellular crude ...

  8. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    Science.gov (United States)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  9. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    Directory of Open Access Journals (Sweden)

    Carrasco Mario

    2012-11-01

    Full Text Available Abstract Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested. All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures.

  10. Phytoplankton extracellular phosphatases: investigation using the ELF (Enzyme Labelled Fluorescence) technique

    Czech Academy of Sciences Publication Activity Database

    Štrojsová, Alena; Vrba, Jaroslav

    2006-01-01

    Roč. 54, č. 4 (2006), s. 715-723 ISSN 1505-2249. [Symposium on European Freshwater Sciences /4./. Krakow, 22.08.2005-26.08.2005] R&D Projects: GA AV ČR(CZ) IAA6017202 Grant - others:FRVŠ(CZ) G4 1841 Institutional research plan: CEZ:AV0Z60170517 Keywords : algae * cyanobacteria * ELF97 phosphate * cell-specific activity Subject RIV: EH - Ecology, Behaviour Impact factor: 0.306, year: 2006

  11. Are bacteria the major producers of extracellular glycolytic enzymes in aquatic environments?

    Czech Academy of Sciences Publication Activity Database

    Vrba, Jaroslav; Callieri, C.; Bittl, T.; Šimek, Karel; Bertoni, R.; Filandr, P.; Hartman, Petr; Hejzlar, Josef; Macek, Miroslav; Nedoma, Jiří

    2004-01-01

    Roč. 89, č. 1 (2004), s. 102-117 ISSN 1434-2944 R&D Projects: GA AV ČR(CZ) IBS6017004; GA ČR(CZ) GA206/99/0028; GA ČR(CZ) GA206/00/0063 Institutional research plan: CEZ:AV0Z6017912 Keywords : ectoenzyme activity * diatoms * Daphnia longispina Subject RIV: DA - Hydrology ; Limnology Impact factor: 0.742, year: 2004

  12. Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders.

    Science.gov (United States)

    Zuccarini, Mariachiara; Giuliani, Patricia; Frinchi, Monica; Mudò, Giuseppa; Serio, Rosa Maria; Belluardo, Natale; Buccella, Silvana; Carluccio, Marzia; Condorelli, Daniele F; Caciagli, Francesco; Ciccarelli, Renata; Di Iorio, Patrizia

    2018-01-01

    Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders.

  13. Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders

    Directory of Open Access Journals (Sweden)

    Mariachiara Zuccarini

    2018-02-01

    Full Text Available Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. Guanosine (GUO and guanine (GUA are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP and guanine deaminase (GDA. Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment. This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory

  14. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    Acid phosphatase; dominance; Drosophila malerkotliana; epigenetics; heterodimeric allozymes; subunit interaction. J. Biosci. | Vol. 27 | No. ... Department of Genetics, Punjab Agricultural University, Ludhiana 141 004, India. *Corresponding ...... the genetic and complementation maps of the locus specifying the enzyme; J.

  15. Vibrio cholerae hemagglutinin(HA)/protease: An extracellular metalloprotease with multiple pathogenic activities.

    Science.gov (United States)

    Benitez, Jorge A; Silva, Anisia J

    2016-06-01

    Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Spatial distribution of enzyme activities in the rhizosphere

    Science.gov (United States)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    The rhizosphere, the tiny zone of soil surrounding roots, certainly represents one of the most dynamic habitat and interfaces on Earth. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. That is why there is an urgent need in spatially explicit methods for the determination of the rhizosphere extension and enzyme distribution. Recently, zymography as a new technique based on diffusion of enzymes through the 1 mm gel plate for analysis has been introduced (Spohn & Kuzyakov, 2013). We developed the zymography technique to visualize the enzyme activities with a higher spatial resolution. For the first time, we aimed at quantitative imaging of enzyme activities as a function of distance from the root tip and the root surface in the soil. We visualized the two dimensional distribution of the activity of three enzymes: β-glucosidase, phosphatase and leucine amino peptidase in the rhizosphere of maize using fluorogenically labelled substrates. Spatial-resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography visualized heterogeneity of enzyme activities along the roots. The activity of all enzymes was the highest at the apical parts of individual roots. Across the roots, the enzyme activities were higher at immediate vicinity of the roots (1.5 mm) and gradually decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

  17. Modification of polymer surfaces to enhance enzyme activity and stability

    DEFF Research Database (Denmark)

    Hoffmann, Christian

    Enzyme immobilization is an important concept for the development of improved biocatalytic processes, primarily through facilitated separation procedures. However, enzyme immobilization usually comes at a price of reduced biocatalytic activity. For this reason, different immobilization methods have...... already been developed, combining the same goal to improve enzyme activity, stability and selectivity. Polymer materials have shown, due to their easy processibility and versatile properties, high potential as enzyme support. However, in order to achieve improved enzyme performance, the combination...... of different factors, such as the nature of the enzyme, the properties of the support, the type of immobilization and the interaction between enzyme and support, has to be taken into consideration. In this thesis, these factors are pursued and addressed by exploiting various types of polymers with focus...

  18. Extracellular cellulolytic enzyme system of Aspergillus japonicus. Pt. 3. Isolation, purification and characterization of multiple forms of endoglucanase

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, R.K.; Dube, Syamalima; Dube, D.K.

    1988-02-01

    The crude culture filtrate of Aspergillus japonicus exhibits marked cellulose and xylan degrading activities. Affinity chromatography of the crude enzyme preparation on concanavalin A (Con A)-Sepharose 4B resolves it into three fractions. The eluted fraction A contains CMCase (CM Case I) and xylanase activities; fraction B shows only CMCase activity (CMCase II); and fraction C exhibits CMCase (CMCase III) and xylanase activities. On further purification by gel filtration, only fraction B imparts a homogeneous preparation that gives a single band on polyacrylamide gel electrophoresis at pH 8.3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of this homogeneous form shows the molecular weight about 57 000 daltons. The first two forms have the least activity toward tamarind kernel polysaccharide (TKP) but the last form, endoglucanase III, is highly reactive to it.

  19. Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host.

    Science.gov (United States)

    Ma, Fang; Guo, Xiao; Fan, Hongjie

    2017-01-15

    bacteremia in its hosts. However, little is known about how S. equi subsp. zooepidemicus interacts with the host innate immune system, particularly innate cells found in the blood. S. equi subsp. zooepidemicus is capable of evading NET-mediated killing via the actions of its potent extracellular nucleases, ENuc and 5Nuc, which directly degrade the NET DNA backbone to deoxyadenosine. In previous studies, other pathogens have required the synergism of nuclease and 5'-nucleotidase to engage in this self-protective process; however, ENuc and 5Nuc both possess nuclease activity and 5'-nucleotidase activity, highlighting the novelty of this discovery. Furthermore, deoxyadenosine impairs phagocytosis but not the intracellular bactericidal activity of macrophages. Here we describe a novel mechanism for S. equi subsp. zooepidemicus extracellular nucleases in NET degradation, which may provide new insights into the pathogen immune evasion mechanism and the prevention and treatment of bacterial disease. Copyright © 2016 American Society for Microbiology.

  20. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

    OpenAIRE

    Hosoe Misa; Takahashi Toru; Ushizawa Koichi; Koshi Katsuo; Kizaki Keiichiro; Mishra Birendra; Sato Takashi; Ito Akira; Hashizume Kazuyoshi

    2010-01-01

    Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. Methods In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and...

  1. Thermodynamic Activity-Based Progress Curve Analysis in Enzyme Kinetics.

    Science.gov (United States)

    Pleiss, Jürgen

    2018-03-01

    Macrokinetic Michaelis-Menten models based on thermodynamic activity provide insights into enzyme kinetics because they separate substrate-enzyme from substrate-solvent interactions. Kinetic parameters are estimated from experimental progress curves of enzyme-catalyzed reactions. Three pitfalls are discussed: deviations between thermodynamic and concentration-based models, product effects on the substrate activity coefficient, and product inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Vps15p regulates the distribution of cup-shaped organelles containing the major eisosome protein Pil1p to the extracellular fraction required for endocytosis of extracellular vesicles carrying metabolic enzymes.

    Science.gov (United States)

    Stein, Kathryn; Winters, Chelsea; Chiang, Hui-Ling

    2017-05-01

    Exosomes are small vesicles secreted from virtually every cell from bacteria to humans. Saccharomyces cerevisiae is a model system to study trafficking of small vesicles in response to changes in the environment. When yeast cells are grown in low glucose, vesicles carrying gluconeogenic enzymes are present as free vesicles and aggregated clusters in the cytoplasm. These vesicles are also secreted into the periplasm and account for more than 90% of total extracellular organelles, while less than 10% are larger 100-300 nm structures with unknown functions. When glucose is added to glucose-starved cells, secreted vesicles are endocytosed and then targeted to the vacuole. Recent secretomic studies indicated that more than 300 proteins involved in diverse biological functions are secreted during glucose starvation and endocytosed during glucose re-feeding. We hypothesised that extracellular vesicles are internalised using novel mechanisms independent of clathrin-mediated endocytosis. Our results showed that vesicles carrying metabolic enzymes were endocytosed at a fast rate, whereas vesicles carrying the heat shock protein Ssa1p were endocytosed at a slow rate. The PI3K regulator Vps15p is critical for the fast internalisation of extracellular vesicles. VPS15 regulates the distribution of the 100-300 nm organelles that contain the major eisosome protein Pil1p to the extracellular fraction. These Pil1p-containing structures were purified and showed unique cup-shape with their centres deeper than the peripheries. In the absence of VPS15, PIL1 or when PIL1 was mutated, the 100-300 nm structures were not observed in the extracellular fraction and the rapid internalisation of vesicles was impaired. We conclude that VPS15 regulates the distribution of the 100-300 nm Pil1p-containing organelles to the extracellular fraction required for fast endocytosis of vesicles carrying metabolic enzymes. This work provides the first evidence showing that Pil1p displayed unique

  3. ORENZA: a web resource for studying ORphan ENZyme activities

    Directory of Open Access Journals (Sweden)

    Labedan Bernard

    2006-10-01

    Full Text Available Abstract Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities and sequence (dataset present in public databases. ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme

  4. ORENZA: a web resource for studying ORphan ENZyme activities.

    Science.gov (United States)

    Lespinet, Olivier; Labedan, Bernard

    2006-10-06

    Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene sequences to the relevant enzymes.

  5. Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis.

    Science.gov (United States)

    Borges, Fernanda Pires; de Brum Vieira, Patrícia; Wiltuschnig, Renata C M; Tasca, Tiana; De Carli, Geraldo Attilio; Bonan, Carla Denise

    2008-06-01

    Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.

  6. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  7. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    International Nuclear Information System (INIS)

    Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

    2006-01-01

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

  8. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  9. Isolation of a tyrosine-activating enzyme from baker's yeast

    NARCIS (Netherlands)

    Ven, A.M. van de; Koningsberger, V.V.; Overbeek, J.Th.G.

    1958-01-01

    The extracts of ether-CO2-frozen baker's yeast contain enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. From the extract a tyrosine-activating enzyme could be isolated, which, judging from ultracentrifugation and electrophoretic data, was about 70% pure

  10. Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions.

    Science.gov (United States)

    Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C

    2017-01-01

    The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

  11. CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells.

    Science.gov (United States)

    Kaur, Sukhbir; Singh, Satya P; Elkahloun, Abdel G; Wu, Weiwei; Abu-Asab, Mones S; Roberts, David D

    2014-07-01

    Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    as homomultimers (Zabin and Villarejo 1975; Kacser and. Burns 1981; Hollacher and Place 1987; Stuber et al 1992;. Xiau et al 1995; Stuber 1999). Using electrophoresis for isolating allozymes [different molecular forms of an enzyme coded by the same gene (Markert 1975)], free from contamination by other allozymes, ...

  13. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  14. Degrading capability and activity of extracellular xylanase secreted ...

    African Journals Online (AJOL)

    Qualitative scanning electron microscopes (SEM) images indicated that after 48 h crude enzymes hydrolysis at 35°C, the material structure was modified. The production of the soluble carbohydrates was up to 2, 400 mg·L-1 for corn straw and 1, 300 mg·L-1 for rice straw. It would hold the potential of further development and ...

  15. Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

    OpenAIRE

    Sinar David Granada García; Antoni Rueda Lorza; Carlos Alberto Peláez

    2014-01-01

    Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual cultur...

  16. Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery.

    Science.gov (United States)

    Nakase, Ikuhiko; Noguchi, Kosuke; Aoki, Ayako; Takatani-Nakase, Tomoka; Fujii, Ikuo; Futaki, Shiroh

    2017-05-16

    Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.

  17. Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

    Science.gov (United States)

    Alonso, Carlos Agustín I; Osycka-Salut, Claudia E; Castellano, Luciana; Cesari, Andreína; Di Siervi, Nicolás; Mutto, Adrián; Johannisson, Anders; Morrell, Jane M; Davio, Carlos; Perez-Martinez, Silvina

    2017-08-01

    Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA

  18. Enzyme hydration, activity and flexibility : A neutron scattering approach

    International Nuclear Information System (INIS)

    Kurkal-Siebert, V.; Finney, J.L.; Daniel, R.M.; Smith, Jeremy C.

    2006-01-01

    Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. The question of whether the hydration-induced enzyme flexibility is important for activity is addressed by performing picosecond dynamic neutron scattering experiments on pig liver esterase powders at various temperatures as well as solutions. At all temperatures and hydrations investigated here, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. At low temperature, increasing hydration resulted in lower flexibility of the enzyme. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The implication of these results is that, although the additional hydration-induced diffusive motion and flexibility at high temperatures in the enzyme detected here may be related to increased activity, they are not required for the enzyme to function

  19. Warming and organic matter sources impact the proportion of dissolved to total activities in marine extracellular enzymatic rates

    KAUST Repository

    Baltar, Federico

    2017-04-19

    Extracellular enzymatic activities (EEAs) are the rate-limiting step in the degradation of organic matter. Extracellular enzymes can be found associated to cells or dissolved in the surrounding water. The proportion of cell-free EEA constitutes in many marine environments more than half of the total activity. This high proportion causes an uncoupling between hydrolysis rates and the actual bacterial activity. However, we do not know what factors control the proportion of dissolved relative to total EEA, nor how this may change in the future ocean. To resolve this, we performed laboratory experiments with water from the Great Barrier Reef (Australia) to study the effects of temperature and dissolved organic matter sources on EEA and the proportion of dissolved EEA. We found that warming increases the rates of organic matter hydrolysis and reduces the proportion of dissolved relative to total EEA. This suggests a potential increase of the coupling between organic matter hydrolysis and heterotrophic activities with increasing ocean temperatures, although strongly dependent on the organic matter substrates available. Our study suggests that local differences in the organic matter composition in tropical coastal ecosystems will strongly affect the proportion of dissolved EEA in response to ocean warming.

  20. Why do crown ethers activate enzymes in organic solvents?

    NARCIS (Netherlands)

    van Unen, D.J.; Engbersen, Johannes F.J.; Reinhoudt, David

    2002-01-01

    One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have

  1. Ligninolytic enzyme activities in mycelium of some wild and ...

    African Journals Online (AJOL)

    Lignin is probably one of the most recalcitrant compounds synthesized by plants. This compound is degraded by few microorganisms. White-rot fungi have been extensively studied due to its powerful ligninolytic enzymes. In this study, ligninolytic enzyme activities of different fungal species (six commercial and 13 wild) were ...

  2. Changes in growth, survival and digestive enzyme activities of Asian ...

    African Journals Online (AJOL)

    A study was conducted to determine the effects of different dietary treatments on the growth, survival and digestive enzyme activities of Mystus nemurus larvae. Newly hatched larvae were reared for 14 days in twelve 15 L glass aquaria (for growth and survival) and eight 300 L fiberglass tanks (for enzyme samples) at a ...

  3. Activity enhancement of ligninolytic enzymes of Trametes versicolor ...

    African Journals Online (AJOL)

    Suspended cultures of white-rot fungus, Trametes versicolor, supplemented with bagasse powder showed a concentration dependent enhancement in the ligninolytic enzymes activity in liquid shake cultures. 2% (w/v) bagasse powder improved greater stability to the enzymes. The optimum pH is 3.5 and the optimum ...

  4. Effect of diffusion on enzyme activity in a microreactor

    NARCIS (Netherlands)

    Swarts, J.W.; Kolfschoten, R.C.; Jansen, M.C.A.A.; Janssen, A.E.M.; Boom, R.M.

    2010-01-01

    To establish general rules for setting up an enzyme microreactor system, we studied the effect of diffusion on enzyme activity in a microreactor. As a model system we used the hydrolysis of ortho-nitrophenyl-ß-d-galactopyranoside by ß-galactosidase from Kluyveromyces lactis. We found that the

  5. Ligninolytic enzyme activities in mycelium of some wild and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-03

    Dec 3, 2008 ... Lignin is probably one of the most recalcitrant compounds synthesized by plants. This compound is degraded by few microorganisms. White-rot fungi have been extensively studied due to its powerful ligninolytic enzymes. In this study, ligninolytic enzyme activities of different fungal species (six commercial ...

  6. General discussion about enzymes activities of radiation injury

    International Nuclear Information System (INIS)

    Vucicevic, M.; Sukalo, I.

    1989-01-01

    Researching reliable and practical indicators of radiation injury, however, is very interesting and considerable department of scientific studies, practical and theoretical. Enzymes activities are among biochemical indicators which are changed after radiation injury. Activity of these specific proteins is important in regulation of every biochemical reaction in existing beings. Biological macromolecules can be damaged by radiation or the cell permeability can be changed. All of these influence directly on enzymes activities. In this paper we present the review of the all important enzymes, indicators of the radiation injury, which variances on reference to normal values are significant of the functional and the structural changes of essential organs (author)

  7. The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

    Directory of Open Access Journals (Sweden)

    Zorana Oreščanin-Dušić

    2018-01-01

    Full Text Available Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

  8. Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.

    Science.gov (United States)

    Simůnek, J; Tishchenko, G; Rozhetsky, K; Bartonová, H; Kopecný, J; Hodrová, B

    2004-01-01

    A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.

  9. Function and biotechnology of extremophilic enzymes in low water activity

    Science.gov (United States)

    2012-01-01

    Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

  10. Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

    Science.gov (United States)

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

    2011-02-17

    Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

  11. Extreme nuclear disproportion and constancy of enzyme activity in a ...

    Indian Academy of Sciences (India)

    Unknown

    Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon of Neurospora crassa. KANDASAMY PITCHAIMANI and RAMESH MAHESHWARI*. Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India. Abstract. Heterokaryons of Neurospora crassa were generated by ...

  12. Changes in photosynthesis and activities of enzymes involved in ...

    African Journals Online (AJOL)

    tolerance, respectively were used to investigate the oxygen consumption rate of photosystem I, the oxygen evolution rate of photosystem II, cab transcript levels, and activities of enzymes involved in photosynthetic carbon reduction cycle.

  13. Enzyme activity measurement via spectral evolution profiling and PARAFAC

    DEFF Research Database (Denmark)

    Baum, Andreas; Meyer, Anne S.; Garcia, Javier Lopez

    2013-01-01

    The recent advances in multi-way analysis provide new solutions to traditional enzyme activity assessment. In the present study enzyme activity has been determined by monitoring spectral changes of substrates and products in real time. The method relies on measurement of distinct spectral...... fingerprints of the reaction mixture at specific time points during the course of the whole enzyme catalyzed reaction and employs multi-way analysis to detect the spectral changes. The methodology is demonstrated by spectral evolution profiling of Fourier Transform Infrared (FTIR) spectral fingerprints using...

  14. Magnetic enzyme membranes as active elements of electrochemical sensors: specific amino acid enzyme elctrodes.

    Science.gov (United States)

    Calvot, C; Berjonneau, A M; Gellf, G; Thomas, D

    1975-11-15

    The basic principle of the described magnetic enzyme electrodes is a kinetic accumulation of CO2 at the active layer electrode interface. The local pCO2 level is linked to three simultaneous phenomena: substrate diffusion in, enzyme reaction CO2 diffusion out. After a transient state there is a stationary state between the quantity of CO2 produced by the enzyme reaction and the CO2 diffusing from the active membrane to the bulk solution. Continuous determination of free amino acids in biological media is useful in biological processing, fermentation, medicine, pharmaceutical industries and biological research. No methods are presently available for any specific continuous measurement of lysine which is of nutritional importance in protein industrial syntheses; of phenylalanine and tyrosine which have to be monitored in several inborn diseases (phenylketonuria being the most important of them); of arginine and histidine which play a still imperfectly understood part in neurochemistry. The use of decarboxylase bearing membranes as sensors in such measurements could offer several novel advantages: (a) a simple device made of a currently manufactured electrode slightly modified by the use of an enzyme membrane; (b) The absence of any enzymic consumption due to the immobilization and the negligible consumption of substrate during the measurements; (c) The sensitivity which can be sharpened by a systematic study of the membrane parameters; (d) the continuous response of the electrode as long as it is in contact with the substrate solution; (e) the further feasibility as a miniature sensor. The magnetic device introduced allows obviously a convenient use of the enzyme electrode, the active part can be removed and replaced without disturbance for the pCO2 electrode itself. The enzyme electrodes are not only useful at the applied point of view but also at the fundamental point of view by allowing a direct measurement of an intra membrane concentration. The influence of

  15. Dietary cholesterol increases paraoxonase 1 enzyme activity

    Science.gov (United States)

    Kim, Daniel S.; Burt, Amber A.; Ranchalis, Jane E.; Richter, Rebecca J.; Marshall, Julieann K.; Nakayama, Karen S.; Jarvik, Ella R.; Eintracht, Jason F.; Rosenthal, Elisabeth A.; Furlong, Clement E.; Jarvik, Gail P.

    2012-01-01

    HDL-associated paraoxonase 1 (PON1) activity has been consistently associated with cardiovascular and other diseases. Vitamins C and E intake have previously been positively associated with PON1 in a subset of the Carotid Lesion Epidemiology and Risk (CLEAR) cohort. The goal of this study was to replicate these findings and determine whether other nutrient intake affected PON1 activity. To predict nutrient and mineral intake values, 1,402 subjects completed a standardized food frequency survey of their dietary habits over the past year. Stepwise regression was used to evaluate dietary and covariate effects on PON1 arylesterase activity. Five dietary components, cholesterol (P < 2.0 × 10−16), alcohol (P = 8.51 × 10−8), vitamin C (P = 7.97 × 10−5), iron (P = 0.0026), and folic acid (0.037) were independently predictive of PON1 activity. Dietary cholesterol was positively associated and predicted 5.5% of PON1 activity, second in variance explained. This study presents a novel finding of dietary cholesterol, iron, and folic acid predicting PON1 activity in humans and confirms prior reported associations, including that with vitamin C. Identifying and understanding environmental factors that affect PON1 activity is necessary to understand its role and that of HDL in human disease. PMID:22896672

  16. Antioxidant Enzyme Activities of some Brassica Species

    Directory of Open Access Journals (Sweden)

    Rodica SOARE

    2017-11-01

    Full Text Available This paper set out to comparatively study five species: white cabbage (Brassica oleracea L. var. capitata alba Alef., red cabbage (Brassica oleracea L. var. capitata f. rubra Alef., Kale (Brassica oleracea L. var. Acephala, cauliflower (Brassica oleracea var. botrytis and broccoli (Brassica oleracea var. cymosa in order to identify those with high enzymatic and antioxidant activities. The enzymatic activity of superoxide dismutase (SOD, catalase (CAT and soluble peroxidase (POX as well as the antioxidant activity against 2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS radical cation were determined. Total superoxide dismutase activity was measured spectrophotometrically based on inhibition in the photochemical reduction of nitroblue tetrazolium. Total soluble peroxidase was assayed by measuring the increase in A436 due to the guaiacol oxidation and the catalase activity was assayed through the colorimetric method. The capacity of extracts to scavenge the ABTS radical cation was assessed colorimetric using Trolox as a standard. The obtained results show that studied enzymatic activities and the antioxidant activity against ABTS vary depending on the analyzed species. So, among the studied Brassicaceae species, it emphasize red cabbage with the highest enzymatic activity (CAT 22.54 mM H2O2/min/g and POX 187.2 mM ΔA/1min/1g f.w. and kale with highest antioxidant activity, of 767 μmol TE/100g f.w. The results of this study recommendintroducing the studied varieties in diet due to the rich antioxidant properties.

  17. Network-Based Methods for Identifying Key Active Proteins in the Extracellular Electron Transfer Process in Shewanella oneidensis MR-1

    OpenAIRE

    Ding, Dewu; Sun, Xiao

    2018-01-01

    Shewanella oneidensis MR-1 can transfer electrons from the intracellular environment to the extracellular space of the cells to reduce the extracellular insoluble electron acceptors (Extracellular Electron Transfer, EET). Benefiting from this EET capability, Shewanella has been widely used in different areas, such as energy production, wastewater treatment, and bioremediation. Genome-wide proteomics data was used to determine the active proteins involved in activating the EET process. We iden...

  18. Kinetic modelling of enzyme inactivation : kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

    NARCIS (Netherlands)

    Schokker, E.P.

    1997-01-01

    The kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F was studied. It was established, by making use of kinetic modelling, that heat inactivation in the temperature range 35 - 70 °C was most likely caused

  19. Occurrence State and Molecular Structure Analysis of Extracellular Proteins with Implications on the Dewaterability of Waste-Activated Sludge.

    Science.gov (United States)

    Wu, Boran; Ni, Bing-Jie; Horvat, Kristine; Song, Liyan; Chai, Xiaoli; Dai, Xiaohu; Mahajan, Devinder

    2017-08-15

    The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R 2 > 0.97, p proteins on the interstitial water removal from WAS.

  20. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation

    NARCIS (Netherlands)

    Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

    2014-01-01

    There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute

  1. Halophilic Bacteria of Lunsu Produce an Array of Industrially Important Enzymes with Salt Tolerant Activity.

    Science.gov (United States)

    Gupta, Sonika; Sharma, Parul; Dev, Kamal; Sourirajan, Anuradha

    2016-01-01

    The halophilic bacterial isolates SS1, SS2, SS3, SS5, and SS8 were characterized for production of industrially important enzymes like amylase, protease, lipase, and glutaminase. Halophilic bacterial isolates SS1 and SS3 exhibited salt dependent extracellular amylase and protease activities. Both the halophilic isolates SS1 and SS3 exhibited maximum amylase and protease activities in the presence of 1.5 and 1.0 M NaCl, respectively, with the optimum pH 8 and temperature 40°C. SS2 showed maximum extracellular protease and lipase activities in the presence of 0.75 M NaCl, at optimum pH of 7, and temperature 37°C. The glutaminase activity of SS3 increased with increase in concentration of NaCl up to 2.5 M. The optimum pH and temperature for L-glutaminase activity of SS3 was 8 and 40°C, respectively. The combined hydrolytic activities of these halophilic bacterial isolates can be used for bioconversion of organic materials to useful products.

  2. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle.

    Science.gov (United States)

    Mishra, Birendra; Kizaki, Keiichiro; Koshi, Katsuo; Ushizawa, Koichi; Takahashi, Toru; Hosoe, Misa; Sato, Takashi; Ito, Akira; Hashizume, Kazuyoshi

    2010-06-11

    Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal

  3. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

    Science.gov (United States)

    2010-01-01

    Background Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. Methods In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. Results EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. Conclusion EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in

  4. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

    Directory of Open Access Journals (Sweden)

    Hosoe Misa

    2010-06-01

    Full Text Available Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. Methods In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. Results EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2 and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. Conclusion EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14

  5. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  6. Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

    Science.gov (United States)

    Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C

    2016-12-01

    Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

  7. Relationship between plasma carotenoids, fruit and vegetable intake, and plasma extracellular superoxide dismutase activity in women: different in health and disease?

    Science.gov (United States)

    Zheng, Jinjin; Rautiainen, Susanne; Morgenstern, Ralf; Wolk, Alicja

    2011-01-01

    Abstract Exogenous antioxidants may influence endogenous antioxidant enzyme activity. We observed in healthy women (n = 95) that higher plasma α-carotene, β-carotene, β-cryptoxanthin, sum of plasma carotenoids, and fruit and vegetable intake were associated with lower plasma extracellular-superoxide dismutase activity. In women with a history of cardiovascular disease, diabetes, or cancer (n = 62), we observed no associations. Our observation that plasma extracellular-superoxide dismutase activity was inversely associated with plasma carotenoids and fruit and vegetable intake in healthy women, but not in women with a history of cardiovascular disease, diabetes, or cancer, suggests that the associations between exogenous and endogenous antioxidants may differ in health and disease.

  8. Enzyme inhibitory activity of selected Philippine plants

    International Nuclear Information System (INIS)

    Sasotona, Joseph S.; Hernandez, Christine C.

    2015-01-01

    In the Philippines, the number one cause of death are cardiovascular diseases. Diseases linked with inflammation are proliferating. This research aims to identify plant extracts that have potential activity of cholesterol-lowering, anti-hypertension, anti-gout, anti-inflammatory and fat blocker agents. Although there are commercially available drugs to treat the aforementioned illnesses, these medicine have adverse side-effects, aside from the fact that they are expensive. The results of this study will serve as added knowledge to contribute to the development of cheaper, more readily available, and effective alternative medicine. 100 plant extracts from different areas in the Philippines have been tested for potential inhibitory activity against Hydroxymethylglutaryl-coenzyme A (HMG-CoA), Lipoxygenase, and Xanthine Oxidase. The plant samples were labeled with codes and distributed to laboratories for blind testing. The effective concentration of the samples tested for Xanthine oxidase is 100 ppm. Samples number 9, 11, 14, 29, 43, 46, and 50 have shown significant inhibitory activity at 78.7%, 78.4%, 70%, 89.2%, 79%, 67.4%, and 67.5% respectively. Samples tested for Lipoxygenase inhibition were set at 33ppm. Samples number 2, 37, 901, 1202, and 1204 have shown significant inhibitory activity at 66, 84.9%, 88.55%, 93.3%, and 84.7% respectively. For HMG-CoA inhibition, the effective concentration of the samples used was 100 ppm. Samples number 1 and 10 showed significant inhibitory activity at 90.1% and 81.8% respectively. (author)

  9. Tissue and plasma enzyme activities in juvenile green iguanas.

    Science.gov (United States)

    Wagner, R A; Wetzel, R

    1999-02-01

    To determine activities of intracellular enzymes in 8 major organs in juvenile green iguanas and to compare tissue and plasma activities. 6 green iguanas iguanas, but high values may not always indicate overt muscle disease. The AMS activity may be specific for the pancreas, but the wide range of plasma activity would likely limit its diagnostic usefulness. Activities of AST and LDH may reflect tissue damage or inflammation, but probably do not reflect damage to specific tissues or organs.

  10. Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes.

    Science.gov (United States)

    Royo, Felix; Palomo, Laura; Mleczko, Justyna; Gonzalez, Esperanza; Alonso, Cristina; Martínez, Ibon; Pérez-Cormenzana, Miriam; Castro, Azucena; Falcon-Perez, Juan M

    2017-02-15

    Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults

  11. Enzyme-polymer composites with high biocatalytic activity and stability

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.; Nauman, E B.; Dordick, Jonathan S.

    2004-08-22

    We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease in activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.

  12. Enzyme activation through the utilization of intrinsic dianion binding energy.

    Science.gov (United States)

    Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

    2017-03-01

    We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

  13. Improving Activity of Salt-Lyophilized Enzymes in Organic Media

    Science.gov (United States)

    Borole, Abhijeet P.; Davison, Brian H.

    Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.

  14. Extraction of Active Enzymes from "Hard-to-Break-Cells"

    DEFF Research Database (Denmark)

    Ottaviani, Alessio; Tesauro, Cinzia; Fjelstrup, S

    We present the utilization of a rolling circle amplification (RCA) based assay to investigate the extraction efficiency of active enzymes from a class of “hard-to-break” cells, yeast Saccaramyces cerevisiae. Current analyses of microorganisms, such as pathogenic bacteria, parasites or particular...... life stages of microorganisms (e.g. spores from bacteria or fungi) is hampered by the lack of efficient lysis protocols that preserve the activity and integrity of the cellular content. Presented herein is a flexible scheme to screen lysis protocols for active enzyme extraction. We also report a gentle...... yet effective approach for extraction of active enzymes by entrapping cells in microdroplets. Combined effort of optimized extraction protocols and effective analytical approaches is expected to generate impact in future disease diagnosis and environmental safety....

  15. Distribution and activity of hydrogenase enzymes in subsurface sediments

    Science.gov (United States)

    Adhikari, R.; Nickel, J.; Glombitza, C.; Spivack, A. J.; D'Hondt, S. L.; Kallmeyer, J.

    2013-12-01

    Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process rather than an overall microbial activity. Since hydrogenase enzymes are intracellular and ubiquitous in subsurface microbial communities, the enzyme activity represents a measure of total activity of the entire microbial community. A hydrogenase activity assay could quantify total metabolic activity without having to identify specific processes. This would be a major advantage in subsurface biosphere studies, where several metabolic processes can occur simultaneously. We quantified hydrogenase enzyme activity and distribution in sediment samples from different aquatic subsurface environments (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico) using a tritium-based assay. We found enzyme activity at all sites and depths. Volumetric hydrogenase activity did not show much variability between sites and sampling depths, whereas cell-specific activity ranged from 10-5 to 1 nmol H2 cell-1 d-1. Activity was lowest in sediment layers where nitrate was detected. Higher activity was associated with samples in which sulfate was the predominant electron acceptor. We found highest activity in samples from environments with >10 ppm methane in the pore water. The results show that cell-specific hydrogenase enzyme activity increases with decreasing energy yield of the electron acceptor used. It is not possible to convert volumetric or cell-specific hydrogenase activity into a

  16. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes.

    Science.gov (United States)

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S -adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  17. Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca2+-Binding Site

    OpenAIRE

    Zeng, Jing; Gao, Xiaowei; Dai, Zheng; Tang, Bing; Tang, Xiao-Feng

    2014-01-01

    Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysi...

  18. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    Directory of Open Access Journals (Sweden)

    De Fine Licht Henrik H

    2012-06-01

    Full Text Available Abstract Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily

  19. Lead action on activity of some enzymes of plants

    International Nuclear Information System (INIS)

    Korolyov, A.N.; Koshkaryova, A.I.

    2008-01-01

    Lead action on activity of some enzymes of young plants of barley double-row (Hordeum distichon L.) families of cereals (Grominea). It is established that activity urease, catalase, ascorbatoxidase is in dependence as from a lead dose in a nutritious solution, and term ontogenesis. At later stages ontogenesis the increase in concentration of lead in an inhabitancy leads to sharp decrease in activity ascorbatoxidase. In the same conditions activity urease and catalase raises.

  20. Early bichemical markers of effects: Enzyme induction, oncogene activation and markers of oxidative damage

    DEFF Research Database (Denmark)

    Poulsen, Henrik E.; Loft, Steffen

    1995-01-01

    Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein......Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein...

  1. Antioxidant enzymes activities in obese Tunisian children

    Directory of Open Access Journals (Sweden)

    Sfar Sonia

    2013-01-01

    Full Text Available Abstract Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls, aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx. Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p  Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response.

  2. Detection of phosphohydrolytic enzyme activity through the oxygen isotope composition of dissolved phosphate

    Science.gov (United States)

    Colman, A. S.

    2016-02-01

    Phosphohydrolytic enzymes play an important role in phosphorus remineralization. As they release phosphate (Pi) from various organophosphorus compounds, these enzymes facilitate the transfer of oxygen atoms from water to the phosphoryl moieties. Most such enzymatic reactions impart a significant isotopic fractionation to the oxygen transferred. If this reaction occurs within a cell, then the resultant oxygen isotope signal is overprinted by continued recycling of the Pi. However, if this reaction occurs extracellularly, then the isotopic signal will be preserved until the Pi is transported back into a cell. Thus, the oxygen isotope composition of Pi (δ18Op) in an aquatic ecosystem can serve as a useful indicator of the mechanisms by which P is remineralized. We develop a time-dependent model illustrating the sensitivity of the δ18O of dissolved phosphate to various modes of P remineralization. The model is informed by cell lysis experiments that reveal the relative proportions of P­i that are directly liberated from cytosol vs. regenerated from co-liberated dissolved organic phosphorus compounds via extracellular hydrolysis. By incorporating both cellular uptake and release fluxes of P, we show that the degree of isotopic disequilibrium in an aquatic ecosystem can be a strong indicator of P remineralization mode. Apparent oxygen isotope equilibrium between Pi and water arises in this model as a steady-state scenario in which fractionation upon cellular uptake of Pi counterbalances the hydrolytic source flux of disequilibrated Pi. Low and high rates of extracellular phosphohydrolase activity are shown to produce steady-state δ18Op values that are respectively above or below thermodynamic equilibrium compositions.

  3. Influence of long-term fertilization on soil enzyme activities

    Directory of Open Access Journals (Sweden)

    Alina Dora SAMUEL

    2009-05-01

    Full Text Available Soil enzyme activities (actual and potential dehydrogenase, catalase, acid and alkaline phosphatase were determined in the 0–10, 10–20, and 20–30 cm layers of a brown luvic soil submitted to a complex fertilization experiment with different types of green manure. It was found that each activity decreased with increasing sampling depth. It should be emphasized that greenmanuring of maize led to a significant increase in each of the five enzymatic activities determined. The enzymatic indicators of soil quality calculated from the values of enzymatic activities showed the order: lupinus + rape + oat > lupinus > vetch + oat + ryegrass > lupinus + oat + vetch > unfertilized plot. This order means that by determination of enzymatic activities valuable information can be obtained regarding fertility status of soils. There were significant correlations of soil enzyme activities with chemical properties.

  4. Early feeding to modify digestive enzyme activity in broiler chickens

    Directory of Open Access Journals (Sweden)

    Milagro León T.

    2014-09-01

    Full Text Available Objective. To evaluate the effect on digestive enzyme activity in broiler chickens by providing food in the first 48 hrs. after birth. Materials and methods. After incubating 300 fertile eggs from Hubbard breeding and immediately after hatching, the chicks were randomly assigned to treatments: fasting (from hatching to 48 hrs.; Hydrated Balanced Food (HBF from birth to 48 hrs.; commercial hydrating supplement (CHS from birth to 48 hrs. The diets were provided ad libitum. After 48 hrs. a commercial diet was fed. At birth and at 48 and 72 hrs. of age 30 chicks/treatment were sacrificed to determine the enzyme activity of maltase, sucrase, alkaline phosphatase, phytase, a-amylase, trypsin and lipase in samples of duodenal or pancreatic homogenate. Results. The supply of HBF or CHS during the first 48 hrs. of life increased the activity of maltase, sucrase and phytase in the first 3 days of life, with values between 1.2 and up to 4-fold compared to the control (p<0.05. Chickens that fasted for the first 48 hrs. had higher activity of the pancreatic enzymes a-amylase, trypsin, and lipase at 72 hrs. of life (p<0.05. Conclusions. The food supply in the first 48 hrs. after hatching increases the duodenal enzyme activity in the intestinal brush border during the first 3 days of age in broiler chickens.

  5. Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland.

    Science.gov (United States)

    González, Débora Alejandra; Barbieri van Haaster, Martín Matías; Quinteros Villarruel, Emmanuel; Brandt, Macarena; Benítez, María Belén; Stranieri, Graciela Mabel; Orman, Betina

    2018-01-01

    Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H 4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    Science.gov (United States)

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  7. Activity of Krebs cycle enzymes in mdx mice.

    Science.gov (United States)

    Comim, Clarissa M; Hoepers, Andreza; Ventura, Letícia; Freiberger, Viviane; Dominguini, Diogo; Mina, Francielle; Mendonça, Bruna P; Scaini, Giselli; Vainzof, Mariz; Streck, Emílio L; Quevedo, João

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a degenerative disease of skeletal, respiratory, and cardiac muscles caused by defects in the dystrophin gene. More recently, brain involvement has been verified. Mitochondrial dysfunction and oxidative stress may underlie the pathophysiology of DMD. In this study we evaluate Krebs cycle enzymes activity in the cerebral cortex, diaphragm, and quadriceps muscles of mdx mice. Cortex, diaphragm, and quadriceps tissues from male dystrophic mdx and control mice were used. We observed increased malate dehydrogenase activity in the cortex; increased malate dehydrogenase and succinate dehydrogenase activities in the diaphragm; and increased citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities in the quadriceps of mdx mice. This study showed increased activity of Krebs cycle enzymes in cortex, quadriceps, and diaphragm in mdx mice. © 2015 Wiley Periodicals, Inc.

  8. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  9. Enzyme activities by indicator of quality in organic soil

    Science.gov (United States)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  10. Free radical scavenging enzymes, activities and their correlation ...

    African Journals Online (AJOL)

    In this study, we investigated erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities as antioxidant enzymes and malondialdehyde (MDA) as a sign of lipid peroxidation inAIH patients, along with routine parameters of liver disease. Present investigations were carried out to evaluate these special parameters ...

  11. Changes in photosynthesis and activities of enzymes involved in ...

    African Journals Online (AJOL)

    AJL

    2012-04-26

    Apr 26, 2012 ... Changes in photosynthesis and activities of enzymes involved in carbon metabolism during exposure ... pigment-protein (cab gene encoding) complexes of PSII. (LHCII), which occupies approximately ... filtered through two layers of Miracloth and the dark green filtrate was centrifuged at 3000 rpm for 5 min ...

  12. Phosphorus fractions, microbial biomass and enzyme activities in ...

    African Journals Online (AJOL)

    Potohar, northern Punjab, Pakistan in September, 2008 and analysed for P fractions and microbial parameters including microbial biomass C, microbial biomass N, microbial biomass P, and activities of dehydrogenase and alkaline phosphatase enzymes. The average size of different P fractions (% of total P) in the soils ...

  13. Comparative Study on Liver Enzymes Activity and Blood Group ...

    African Journals Online (AJOL)

    The aim of this study is to determine the activities of some selected liver enzymes amongst apparently healthy subjects of different blood groups. The study involved 95 apparently healthy students of Ambrose Alli University, Ekpoma, Edo State, Nigeria, between the ages of 18-30, and distributed as follows; blood group O ...

  14. Physicochemical Properties and Enzymes Activity Studies in a ...

    African Journals Online (AJOL)

    JTEkanem

    2009-11-04

    Nov 4, 2009 ... Soil Nitrogen (N),. Phosphorus (P), Calcium (Ca) and Magnesium (Mg) were also elevated in contaminated soil. These results suggest that the soil is not yet suitable for agricultural activity. Keywords: Refined-Oil Spill, Soil Enzymes, Physicochemical Properties, Eluama. *To whom correspondence should ...

  15. Modulation of Antioxidant Enzyme Expression and Activity by ...

    African Journals Online (AJOL)

    Renal toxicity produced by paraquat involves the generation of reactive oxygen species (ROS) which can overwhelm antioxidant defences, leading to oxidant injury. However, there are conflicting reports regarding the activity and/or expression of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) ...

  16. Modulation of enzyme activities following the coadministration of ...

    African Journals Online (AJOL)

    It could be inferred from the results therefore that the intrinsic properties of chemical substances could be modulated or modified intracellularly when in interaction with other compounds and even with the cell system. Keywords: Food additives, Chloroquine, Potassium bromate, Co-administration, Enzyme activity, Modulation ...

  17. Effect of extracellular adenosine triphosphate on activity of osteoblast like cells - biomed 2013.

    Science.gov (United States)

    Mehta, Siddhant K; Tucci, Michelle A; Benghuzzi, Hamed A

    2013-01-01

    Platelet dense granules contain serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP). These molecules are present in platelet rich plasma (PRP), and may therefore have an impact on the efficacy of PRP therapy. Additionally, nucleotides are important extracellular signaling molecules in a variety of tissue types including bone. The purpose of this investigation was to evaluate the in vitro dose-dependent effects of extracellular adenosine triphosphate (ATP) exposure on activity of human osteoblast-like cells. MG-63 cells were exposed to phosphate buffered saline (control group) or ATP solution (20 µM, 100µM, 200 µM). Osteoblast viability was evaluated at 24, 48, and 72 hours using nonspecific and osteoblast-specific markers and cellular morphology. No significant differences in total protein, malonlydialdehyde (MDA), or glutathione were observed with ATP exposure at any timepoint. High dose ATP exposure resulted in a significantly higher production of nitric oxide compared to controls and other groups. With respect to alkaline phosphatase activity and osteopontin production, no significant differences were present with ATP exposure. Overall conclusion: Extracellular ATP exposure modulated osteoblast activity with no change in cell viability in vitro.

  18. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  19. A metal-based inhibitor of NEDD8-activating enzyme.

    Directory of Open Access Journals (Sweden)

    Hai-Jing Zhong

    Full Text Available A cyclometallated rhodium(III complex [Rh(ppy(2(dppz](+ (1 (where ppy=2-phenylpyridine and dppz=dipyrido[3,2-a:2',3'-c]phenazine dipyridophenazine has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE. The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.

  20. Inhibitors of Testosterone Biosynthetic and Metabolic Activation Enzymes

    Directory of Open Access Journals (Sweden)

    Leping Ye

    2011-12-01

    Full Text Available The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1 for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B, for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1 for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3 for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1 and 2 (SRD5A2 in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz and plant constituents (genistein and gossypol. This paper reviews these endocrine disruptors targeting steroidogenic enzymes.

  1. Inhibitors of testosterone biosynthetic and metabolic activation enzymes.

    Science.gov (United States)

    Ye, Leping; Su, Zhi-Jian; Ge, Ren-Shan

    2011-12-02

    The Leydig cells of the testis have the capacity to biosynthesize testosterone from cholesterol. Testosterone and its metabolically activated product dihydrotestosterone are critical for the development of male reproductive system and spermatogenesis. At least four steroidogenic enzymes are involved in testosterone biosynthesis: Cholesterol side chain cleavage enzyme (CYP11A1) for the conversion of cholesterol into pregnenolone within the mitochondria, 3β-hydroxysteroid dehydrogenase (HSD3B), for the conversion of pregnenolone into progesterone, 17α-hydroxylase/17,20-lyase (CYP17A1) for the conversion of progesterone into androstenedione and 17β-hydroxysteroid dehydrogenase (HSD17B3) for the formation of testosterone from androstenedione. Testosterone is also metabolically activated into more potent androgen dihydrotestosterone by two isoforms 5α-reductase 1 (SRD5A1) and 2 (SRD5A2) in Leydig cells and peripheral tissues. Many endocrine disruptors act as antiandrogens via directly inhibiting one or more enzymes for testosterone biosynthesis and metabolic activation. These chemicals include industrial materials (perfluoroalkyl compounds, phthalates, bisphenol A and benzophenone) and pesticides/biocides (methoxychlor, organotins, 1,2-dibromo-3-chloropropane and prochloraz) and plant constituents (genistein and gossypol). This paper reviews these endocrine disruptors targeting steroidogenic enzymes.

  2. Co-ordinate activation of lipogenic enzymes in hepatocellular carcinoma.

    Science.gov (United States)

    Yahagi, Naoya; Shimano, Hitoshi; Hasegawa, Kiyoshi; Ohashi, Kenichi; Matsuzaka, Takashi; Najima, Yuho; Sekiya, Motohiro; Tomita, Sachiko; Okazaki, Hiroaki; Tamura, Yoshiaki; Iizuka, Yoko; Ohashi, Ken; Nagai, Ryozo; Ishibashi, Shun; Kadowaki, Takashi; Makuuchi, Masatoshi; Ohnishi, Shin; Osuga, Jun-ichi; Yamada, Nobuhiro

    2005-06-01

    Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent. Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-differentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in part on the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level. We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes, such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue. In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved in this activation.

  3. Extracellular ATP modulates synaptic plasticity induced by activation of metabotropic glutamate receptors in the hippocampus.

    Science.gov (United States)

    Yamazaki, Yoshihiko; Fujii, Satoshi

    2015-01-01

    Synaptic plasticity is believed to be a cellular mechanism for memory formation in the brain. It has been known that the metabotropic glutamate receptor (mGluR) is required for persistent forms of memory and induction of synaptic plasticity. Application of mGluR agonists induces synaptic plasticity in the absence of electrical conditioning stimulation, such as high or low frequency stimulation. The direction of the mGluR-induced synaptic plasticity, i.e., either long-term potentiation (LTP) or long-term-depression (LTD), is dependent on whether N-methyl-D-aspartate receptors (NMDARs) are co-activated with mGluRs. ATP has modulatory effects on neuronal functions and, in particular, there is increasing evidence that it plays a crucial role in synaptic plasticity. LTP can be induced by application of ATP, and this effect is inhibited by NMDAR antagonist. Although cooperative effects of NMDARs and mGluRs and of NMDARs and extracellular ATP in synaptic plasticity have been revealed, the effect of extracellular ATP on mGluR-induced synaptic plasticity is unknown. In this article, we summarize published data on mGluR- and ATP-induced synaptic plasticity, and present new data showing that extracellular ATP facilitates both the LTP and LTD induced by mGluR activation.

  4. ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

    Science.gov (United States)

    Sehgal, Pankaj; Olesen, Claus; Møller, Jesper V

    2016-01-01

    Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

  5. Microfluidics-Enabled Enzyme Activity Measurement in Single Cells.

    Science.gov (United States)

    Tesauro, Cinzia; Frøhlich, Rikke; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R

    2015-01-01

    Cellular heterogeneity has presented a significant challenge in the studies of biology. While most of our understanding is based on the analysis of ensemble average, individual cells may process information and respond to perturbations very differently. Presented here is a highly sensitive platform capable of measuring enzymatic activity at the single-cell level. The strategy innovatively combines a rolling circle-enhanced enzyme activity detection (REEAD) assay with droplet microfluidics. The single-molecule sensitivity of REEAD allows highly sensitive detection of enzymatic activities, i.e. at the single catalytic event level, whereas the microfluidics enables isolation of single cells. Further, confined reactions in picoliter-sized droplets significantly improve enzyme extraction from human cells or microorganisms and result in faster reaction kinetics. Taken together, the described protocol is expected to open up new possibilities in the single-cell research, particularly for the elucidation of heterogeneity in a population of cells.

  6. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  7. Effect of Barley and Enzyme on Performance, Carcass, Enzyme Activity and Digestion Parameters of Broilers

    Directory of Open Access Journals (Sweden)

    majid kalantar

    2016-04-01

    Full Text Available Introduction Corn has been recently used for producing ethanol fuel in the major corn-producing countries such as the US and Brazil. Recent diversion of corn for biofuel production along with the increased world's demand for this feedstuff has resulted in unprecedented rise in feed cost for poultry worldwide. Alternative grains such as wheat and barley can be successfully replaced for corn in poultry diets. These cereal grains can locally grow in many parts of the world as they have remarkably lower water requirement than corn. Wheat and barley are generally used as major sources of energy in poultry diets. Though the major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls (Olukosi et al. 2007; Mirzaie et al. 2012. NSPs are generally considered as anti-nutritional factors (Jamroz et al. 2002. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value (Olukosi et al. 2007.Wheat and barley are generally used as major sources of energy in poultry diets. The major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls. NSPs are generally considered as anti-nutritional factors. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value. The major part of NSPs in barley comprises polymers of (1→3 (1→4-β- glucans which could impede growth factors and consequently carcass quality through lowering the rate and amount of available nutrients in the mucosal surface of the intestinal. Materials and Methods This experiment was conducted to evaluate the effect of corn and barley based diets supplemented with multi-enzyme on growth, carcass, pancreas enzyme activity and physiological characteristics of broilers. A total number of 375 one day old

  8. Extracellular metalloproteinases in Phytomonas serpens.

    Science.gov (United States)

    Vermelho, Alane B; Almeida, Flávia V S; Bronzato, Leandro S; Branquinha, Marta H

    2003-03-01

    The detection of extracellular proteinases in Phytomonas serpens, a trypanosomatid isolated from tomato fruits, is demonstrated in this paper. Maximal production occurred at the end of the logarithmic phase of growth. These enzymes exhibited selective substrate utilization in SDS-PAGE, being more active with gelatin; hemoglobin and bovine serum albumin were not degraded. Three proteinases were detected in SDS-PAGE-gelatin, with apparent molecular masses between 94 and 70 kDa. The proteolytic activity was completely blocked by 1,10-phenanthroline and strongly inhibited by EDTA, whereas a partial inhibition was observed with trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) and soybean trypsin inhibitor; phenylmethylsulfonyl fluoride weakly inhibited the enzymes. This inhibition profile indicated that these extracellular proteinases belong to the metalloproteinase class.

  9. Degradation of olive mill wastewater by the induced extracellular ligninolytic enzymes of two wood-rot fungi.

    Science.gov (United States)

    Zerva, Anastasia; Zervakis, Georgios I; Christakopoulos, Paul; Topakas, Evangelos

    2017-12-01

    Olive mill wastewater (OMWW) is a major problem in olive oil - producing countries, due to its high organic load and concentration in phenols that are toxic for marine life, plants and soil microorganisms. In the present study, two mushroom species were tested in regard to their OMWW's oxidative capacity, Pleurotus citrinopileatus LGAM 28684 and Irpex lacteus LGAM 238. OMWW (25% v/v) degradation was investigated for several culture conditions, namely pH, agitation speed, nitrogen-based supplements and their concentration. The selected values were pH 6, agitation rate 150 rpm, 30 g L -1 corn steep liquor as nitrogen source for P. citrinopileatus and 20 g L -1 diammonium tartrate for I. lacteus. The two strains performed well in cultures supplemented with OMWW, generating very high titers of oxidative enzymes and achieving more than 90% color and phenols reduction within a 24 days cultivation period. In addition, the amount of glucans present in the fungal biomass was assessed. Hence, P. citrinopileatus and I. lacteus appear as potent degraders of OMWW with the ability to use the effluent as a substrate for the production of biotechnologically important enzymes and valuable fungal glucans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  11. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    Science.gov (United States)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  12. Growth characteristics and enzyme activity in Batrachochytrium dendrobatidis isolates.

    Science.gov (United States)

    Symonds, E Pearl; Trott, Darren J; Bird, Philip S; Mills, Paul

    2008-09-01

    Batrachochytrium dendrobatidis is a member of the phylum Chytridiomycota and the causative organism chytridiomycosis, a disease of amphibians associated with global population declines and mass mortality events. The organism targets keratin-forming epithelium in adult and larval amphibians, which suggests that keratinolytic activity may be required to infect amphibian hosts. To investigate this hypothesis, we tested 10 isolates of B. dendrobatidis for their ability to grow on a range of keratin-supplemented agars and measured keratolytic enzyme activity using a commercially available kit (bioMerieux API ZYM). The most dense and fastest growth of isolates were recorded on tryptone agar, followed by growth on frog skin agar and the slowest growth recorded on feather meal and boiled snake skin agar. Growth patterns were distinctive for each nutrient source. All 10 isolates were strongly positive for a range of proteolytic enzymes which may be keratinolytic, including trypsin and chymotrypsin. These findings support the predilection of B. dendrobatidis for amphibian skin.

  13. Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors

    Science.gov (United States)

    2012-01-01

    Background The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2. Results PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3), laminarin, or piceatannol (a spleen tyrosine kinase inhibitor), suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4). PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression. Conclusions Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α. PMID:22883599

  14. Rapid Shifts in Soil Nutrients and Decomposition Enzyme Activity in Early Succession Following Forest Fire

    Directory of Open Access Journals (Sweden)

    Joseph E. Knelman

    2017-09-01

    Full Text Available While past research has studied forest succession on decadal timescales, ecosystem responses to rapid shifts in nutrient dynamics within the first months to years of succession after fire (e.g., carbon (C burn-off, a pulse in inorganic nitrogen (N, accumulation of organic matter, etc. have been less well documented. This work reveals how rapid shifts in nutrient availability associated with fire disturbance may drive changes in soil enzyme activity on short timescales in forest secondary succession. In this study, we evaluate soil chemistry and decomposition extracellular enzyme activity (EEA across time to determine whether rapid shifts in nutrient availability (1–29 months after fire might control microbial enzyme activity. We found that, with advancing succession, soil nutrients correlate with C-targeting β-1,4-glucosidase (BG EEA four months after the fire, and with N-targeting β-1,4-N-acetylglucosaminidase (NAG EEA at 29 months after the fire, indicating shifting nutrient limitation and decomposition dynamics. We also observed increases in BG:NAG ratios over 29 months in these recently burned soils, suggesting relative increases in microbial activity around C-cycling and C-acquisition. These successional dynamics were unique from seasonal changes we observed in unburned, forested reference soils. Our work demonstrates how EEA may shift even within the first months to years of ecosystem succession alongside common patterns of post-fire nutrient availability. Thus, this work emphasizes that nutrient dynamics in the earliest stages of forest secondary succession are important for understanding rates of C and N cycling and ecosystem development.

  15. Partial Characterization of α-Galactosidic Activity from the Antarctic Bacterial Isolate, . LX-20 as a Potential Feed Enzyme Source

    Directory of Open Access Journals (Sweden)

    Inkyung Park

    2012-06-01

    Full Text Available An Antarctic bacterial isolate displaying extracellular α-galactosidic activity was named Paenibacillus sp. LX-20 based on 16S rRNA gene sequence analysis. Optimal activity for the LX-20 α-galactosidase occurred at pH 6.0–6.5 and 45°C. The enzyme immobilized on the smart polymer Eudragit L-100 retained 70% of its original activity after incubation for 30 min at 50°C, while the free enzyme retained 58% of activity. The enzyme had relatively high specificity for α-D-galactosides such as p-nitrophenyl-α-galactopyranoside, melibiose, raffinose and stachyose, and was resistant to some proteases such as trypsin, pancreatin and pronase. Enzyme activity was almost completely inhibited by Ag+, Hg2+, Cu2+, and sodium dodecyl sulfate, but activity was not affected by β-mercaptoethanol or EDTA. LX-20 α-galactosidase may be potentially useful as an additive for soybean processing in the feed industry.

  16. Evaluation of pancreatin stability through enzyme activity determination

    Directory of Open Access Journals (Sweden)

    Terra Gleysson De Paula

    2016-09-01

    Full Text Available Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity.

  17. In vivo enzyme activity in inborn errors of metabolism

    International Nuclear Information System (INIS)

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D.

    1990-01-01

    Low-dose continuous infusions of [2H5]phenylalanine, [1-13C]propionate, and [1-13C]leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD

  18. In vivo enzyme activity in inborn errors of metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. (Clinical Research Centre, Harrow (England))

    1990-08-01

    Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

  19. Detection of extracellular protease activity in different species and genera of ectomycorrhizal fungi.

    Science.gov (United States)

    Nygren, Cajsa M R; Edqvist, Johan; Elfstrand, Malin; Heller, Gregory; Taylor, Andy F S

    2007-05-01

    In northern forest ecosystems, most soil nitrogen (N) is in organic form and forest trees are largely dependent on ectomycorrhizal (ECM) fungi and their degradative abilities for N uptake. The ability of ECM fungi to acquire N from organic substrates should, therefore, be a widespread trait given its ecological importance. However, little is known about the degradative abilities of most ECM fungi as they remain untested due to problems of isolation or extremely slow growth in pure culture. In this paper, we present data on extracellular protease activity of 32 species of ECM fungi, most of which have not previously been cultured. Milk powder plates and zymograms were compared for detecting protease activity in these intractable species. In total, 29/32 of the species produced extracellular protease activity, but detection was method dependent. Growth on milk powder plates detected protease activity in 28 of 32 species, while zymograms only detected proteases in Amanita muscaria, Russula chloroides, Lactarius deterrimus and Lactarius quieticolor. The study supports the hypothesis that protease excretion is a widespread physiological trait in ECM fungi and that this ability is of considerable significance for nitrogen uptake in forest ecosystems.

  20. Lignocellulose-converting enzyme activity profiles correlate with molecular systematics and phylogeny grouping in the incoherent genus Phlebia (Polyporales, Basidiomycota).

    Science.gov (United States)

    Kuuskeri, Jaana; Mäkelä, Miia R; Isotalo, Jarkko; Oksanen, Ilona; Lundell, Taina

    2015-10-19

    The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species. Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes. Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates. Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.

  1. Extracellular acidosis activates ASIC-like channels in freshly isolated cerebral artery smooth muscle cells.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Swenson, Alyssa; Barnard, John M; Hamilton, Gina; Chiposi, Rumbidzayi; Drummond, Heather A

    2010-05-01

    Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular H(+)-induced inward currents in 46% of cells tested (n = 58 of 126 VSMCs, pH 6.5-5.0). The percentage of responsive cells and the current amplitude increased as the external H(+) concentration increased (pH(6.0), n = 28/65 VSMCs responsive, mean current density = 8.1 +/- 1.2 pA/pF). Extracellular acidosis (pH(6.0)) shifted the whole cell reversal potential toward the Nernst potential of Na(+) (n = 6) and substitution of extracellular Na(+) by N-methyl-d-glucamine abolished the inward current (n = 6), indicating that Na(+) is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 microM) inhibited proton-induced currents to 16.5 +/- 8.7% of control (n = 6, pH(6.0)). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTx1 either 1) abolished H(+)-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of H(+)-induced currents (76%, 34/45), or 3) failed to promote H(+) activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b.

  2. Enzyme activities and microbial biomass in topsoil layer during spontaneous succession in spoil heaps after brown coal mining

    Energy Technology Data Exchange (ETDEWEB)

    Baldrian, P.; Trogl, J.; Frouz, J.; Snajdr, J.; Valaskova, V.; Merhautova, V.; Cajthaml, T.; Herinkova, J. [ASCR, Prague (Czech Republic). Institute for Microbiology

    2008-09-15

    Changes in the activity of extracellular enzymes (cellobiohydrolase, beta-glucosidase, beta-xylosidase, chitinase, arylsulfatase and phosphatases) and the changes in microbial community and abiotic properties in the topsoil layer, as well as soil abiotic properties during primary succession were investigated in a brown coal mine deposit area near Sokolov, Czech Republic. The study considered the chronosequence of 4 post-mining plots, 4-, 12-, 21 - and 45-year old. The 4-year old site had no vegetation cover. Herbs and grasses (mainly Calamagrostis epigeios) were present on the 12-year old plot, shrubs (Salix caprea) occurred on the 21-year old plot and tree cover (Betula spp. and Populus tremuloides) developed on the 45-year old plot. Soil pH gradually decreased with site age, while the content of K, C and N peaked in the 21-year old site, being significantly lower in the 45-year old site and much lower in the 4- and 12-year old sites. Phosphatase activities were strongly affected by seasonality while the activities of all the other enzymes measured were more influenced by the effects of succession age and soil layer than by seasonality. Succession age was also the most important factor affecting the total and bacterial PLFA contents, followed by the effects of soil layer and season while for the fungal biomass content-related properties (ergosterol, fungal PLFA and the fungal/bacterial PLFA ratio), season was the most important. Activities of individual enzymes in the topsoil (0-5 cm depth) were significantly affected by both site age and season. Cellobiohydrolase and beta-xylosidase were more affected by site age while chitinase and phosphatases were more affected by season. Enzyme activity increased with succession age. Comparison of the effect of site and season on enzyme activity showed that season played a principal role in the enzyme activity of the entire 0-5 cm component of topsoil, as well the soil layers when evaluated separately.

  3. Stromal cell extracellular vesicular cargo mediated regulation of breast cancer cell metastasis via ubiquitin conjugating enzyme E2 N pathway.

    Science.gov (United States)

    Vallabhaneni, Krishna C; Penfornis, Patrice; Xing, Fei; Hassler, Yoni; Adams, Kristen V; Mo, Yin-Yuan; Watabe, Kounosuke; Pochampally, Radhika

    2017-12-15

    Mesenchymal stromal cells (hMSCs) have been used to understand the stromal cell properties in solid tumors because of their ablity to differentiate into most cell types. We investigated the role of EVs from hMSCs (hMSC-EVs) in breast cancer metastasis using MDA-MB-231 parental cell line and organotropic sub-lines. We demonstrated that hMSC-EVs significantly suppressed the metastatic potential of the parental cell line when compared to their organotropic sublines. hMSC-EVs induce dormancy in the parental cell line but not in their organotropic sub-lines and miR-205 and miR-31 from EV cargo played a role. Further, Ubiquitin Conjugating Enzyme E2 N (UBE2N/Ubc13) - metastasis-regulating gene, is a target of these miRNAs and silencing of UBE2N/Ubc13 expression significantly suppressed migration, invasion, and proliferation of breast cancer cells. To summarize, hMSC-EVs support primary breast tumor progression but suppress the metastasis of breast cancer cells that are not organ-committed through the UBE2N/Ubc13 pathway and play a role in premetastic niche formation.

  4. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    Science.gov (United States)

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  5. Optimization of extracellular catalase production from Aspergillus ...

    African Journals Online (AJOL)

    The studies of the effect of each variable and the establishment of a correlation between the response of enzyme activity and variables revealed that the link is a multiple linear regression form. The optimization was carried out through a simplex algorithm. The amount of extracellular catalase produced by the strain in the ...

  6. Calcium-protein interactions in the extracellular environment: calcium binding, activation, and immunolocalization of a collagenase/gelatinase activity expressed in the sea urchin embryo.

    Science.gov (United States)

    Mayne, J; Robinson, J J

    1998-12-15

    We have purified and characterized a collagenase/gelatinase activity expressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymography revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect could be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred with an apparent kd value of 3.7 mM. Two modes of calcium binding to the 41-kDa subunit were detected; up to 80 moles of calcium bound with a kd value of 0.5 mM, while an additional 120 moles bound with a kd value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mol, indicating the availability of substantial numbers of weak Ca2+-binding sites. Calcium binding did not result in either secondary or quaternary structural changes in the collagenase/gelatinase, suggesting that Ca2+ may facilitate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in blastocoelic fluid and in the hyalin fraction dissociated from 1-h-old embryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed throughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina, respectively.

  7. ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2014-02-01

    Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 U.mg-1 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 U.mg-1 protein.

  8. Relationship between Estradiol and Antioxidant Enzymes Activity of Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Nasrin Sheikh

    2009-01-01

    Full Text Available Some evidence suggests the neuroprotection of estrogen provided by the antioxidant activity of this compound. The main objective of this study was to determine the level of estradiol and its correlation with the activity of antioxidant enzymes, total antioxidant status and ferritin from ischemic stroke subjects. The study population consisted of 30 patients with acute ischemic stroke and 30 controls. There was no significant difference between estradiol in stroke and control group. The activity of superoxide dismutase and level of ferritin was higher in stroke compared with control group (<.05, <.001, resp.. There was no significant correlation between estradiol and glutathione peroxidase, glutathione reductase, catalase, total antioxidant status, and ferritin in stroke and control groups. We observed inverse correlation between estradiol with superoxide dismutase in males of stroke patients (=−0.54, =.029. Our results supported that endogenous estradiol of elderly men and women of stroke or control group has no antioxidant activity.

  9. ANTIOXIDANT ENZYME ACTIVITY AND FRESH-CUT ARRACACHA QUALITY

    Directory of Open Access Journals (Sweden)

    Hêmina Carla Vilela

    2015-06-01

    Full Text Available The arracacha is an alternative of fresh-cut product; however it can be easily degraded after the processing techniques. The objective of this work was to evaluate the useful life of fresh-cut arracacha submitted to two types of cuts and storage, as well as to evaluate the activity of antioxidant enzymes. The roots were selected, sanitized and submitted to two cut types: cubed and grated. Then they were evaluated at 3 times: 0, 3 and 7 days. The cutting in cubes provided higher quality and lower SOD, CAT and APX activity. However, the grated product presented higher PG activity and lower PPO activity. The microbiological safety and the nutritional value were maintained in both cuts during the whole storage period. The useful life, regarding the physicochemical, nutritional and microbiological aspects, can be established at 7 days under refrigeration for fresh-cut arracacha.

  10. Increased resiliency and activity of microbial mediated carbon cycling enzymes in diversified bioenergy cropping systems

    Science.gov (United States)

    Upton, R.; Bach, E.; Hofmockel, K. S.

    2017-12-01

    Microbes are mediators of soil carbon (C) and are influenced in membership and activity by nitrogen (N) fertilization and inter-annual abiotic factors. Microbial communities and their extracellular enzyme activities (EEA) are important parameters that influence ecosystem C cycling properties and are often included in microbial explicit C cycling models. In an effort to generate model relevant, empirical findings, we investigated how both microbial community structure and C degrading enzyme activity are influenced by inter-annual variability and N inputs in bioenergy crops. Our study was performed at the Comparison of Biofuel Systems field-site from 2011 to 2014, in three bioenergy cropping systems, continuous corn (CC) and two restored prairies, both fertilized (FP) and unfertilized (P). We hypothesized microbial community structure would diverge during the prairie restoration, leading to changes in C cycling enzymes over time. Using a sequencing approach (16S and ITS) we determined the bacterial and fungal community structure response to the cropping system, fertilization, and inter-annual variability. Additionally, we used EEA of β-glucosidase, cellobiohydrolase, and β-xylosidase to determine inter-annual and ecosystem impacts on microbial activity. Our results show cropping system was a main effect for microbial community structure, with corn diverging from both prairies to be less diverse. Inter-annual changes showed that a drought occurring in 2012 significantly impacted microbial community structure in both the P and CC, decreasing microbial richness. However, FP increased in microbial richness, suggesting the application of N increased resiliency to drought. Similarly, the only year in which C cycling enzymes were impacted by ecosystem was 2012, with FP supporting higher potential enzymatic activity then CC and P. The highest EEA across all ecosystems occurred in 2014, suggesting the continued root biomass and litter build-up in this no till system

  11. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    Science.gov (United States)

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs.

  12. Histochemical location of key enzyme activities involved in receptivity and self-incompatibility in the olive tree (Olea europaea L.).

    Science.gov (United States)

    Serrano, Irene; Olmedilla, Adela

    2012-12-01

    Stigma-surface and style enzymes are important for pollen reception, selection and germination. This report deals with the histochemical location of the activity of four basic types of enzyme involved in these processes in the olive (Olea europaea L.). The detection of peroxidase, esterase and acid-phosphatase activities at the surface of the stigma provided evidence of early receptivity in olive pistils. The stigma maintained its receptivity until the arrival of pollen. Acid-phosphatase activity appeared in the style at the moment of anthesis and continued until the fertilization of the ovule. RNase activity was detected in the extracellular matrix of the styles of flowers just before pollination and became especially evident in pistils after self-pollination. This activity gradually decreased until it practically disappeared in more advanced stages. RNase activity was also detected in pollen tubes growing in pollinated pistils and appeared after in vitro germination in the presence of self-incompatible pistils. These findings suggest that RNases may well be involved in intraspecific pollen rejection in olive flowers. To the best of our knowledge this is the first time that evidence of enzyme activity in stigma receptivity and pollen selection has been described in this species. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  13. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

    Science.gov (United States)

    Thålin, Charlotte; Daleskog, Maud; Göransson, Sophie Paues; Schatzberg, Daphne; Lasselin, Julie; Laska, Ann-Charlotte; Kallner, Anders; Helleday, Thomas; Wallén, Håkan; Demers, Mélanie

    2017-06-01

    There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

  14. Virtual Biochemistry – pH effect on enzyme activity

    Directory of Open Access Journals (Sweden)

    D.N. Heidrich

    2011-04-01

    Full Text Available Protocols of laboratory experiments, followed by teacher's explanation, not always clearly translate to the student the dynamics to beadopted for the implementation of the proposed practice. One of these cases is related to the study of the effect of pH on enzyme activity. For better help the understanding of the technical procedure, a hypermedia was built based on a protocol adopted at the Department of Biochemistry, UFSC. The hypermedia shows how theeffect of variations in pH can be observed  in vitro. Taking as example salivary amylase and the consumption of starch (substrate by means of iodine staining, a set of pH buffers was tested to identify the best pH for this enzyme  activity. This hypermedia as introductory tool for such practice was tested on aNutrition course classroom. Students agree that the hypermedia provided a better understanding of the proposed activities. Teachers also notice a smallerreagents consumption and reduction of the time spent by the students in the achievement of the experiment.

  15. Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors

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    Wang Cheng-Li

    2012-08-01

    Full Text Available Abstract Background The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2. Results PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3, laminarin, or piceatannol (a spleen tyrosine kinase inhibitor, suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4. PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression. Conclusions Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α.

  16. Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells.

    Science.gov (United States)

    Jong, Ambrose Y; Wu, Chun-Hua; Li, Jingbo; Sun, Jianping; Fabbri, Muller; Wayne, Alan S; Seeger, Robert C

    2017-01-01

    Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations : NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron

  17. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    Science.gov (United States)

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

    KAUST Repository

    Suen, KinMan

    2013-05-01

    Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

  19. Effects of chemical inhibitors and apyrase enzyme further document a role for apyrases and extracellular ATP in the opening and closing of stomates in Arabidopsis.

    Science.gov (United States)

    Clark, Greg; Darwin, Cameron; Mehta, Viraj; Jackobs, Faith; Perry, Tyler; Hougaard, Katia; Roux, Stan

    2013-11-01

    In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results further confirm the role of extracellular ATP in regulating stomatal apertures.

  20. Climate and root proximity as dominant drivers of enzyme activity and C and N isotopic signature in soil

    Science.gov (United States)

    Stock, Svenja; Köster, Moritz; Dippold, Michaela; Boy, Jens; Matus, Francisco; Merino, Carolina; Nájera, Francisco; Spielvogel, Sandra; Gorbushina, Anna; Kuzyakov, Yakov

    2017-04-01

    The Chilean ecosystems provide a unique study area to investigate biotic controls on soil organic matter (SOM) decomposition and mineral weathering depending on climate (from hyper arid to temperate humid). Microorganisms play a crucial role in the SOM decomposition, nutrient release and cycling. By means of extracellular enzymes microorganisms break down organic compounds and provide nutrients for plants. Soil moisture (abiotic factor) and root carbon (biotic factor providing easily available energy source for microorganisms), are important factors for microbial decomposition of SOM and show strong gradients along the investigated climatic gradient. A high input of root carbon increases microbial activity and enzyme production, and facilitates SOM breakdown and nutrient release The aim of this study was to determine the potential enzymatic SOM decomposition and nutrient release depending on root proximity and precipitation. C and N contents, δ13C and δ15N values, and kinetics (Vmax, Km) of six extracellular enzymes, responsible for C, N, and P cycles, were quantified in vertical (soil depth) and horizontal (from roots to bulk soil) gradients in two climatic regions: within a humid temperate forest and a semiarid open forest. The greater productivity of the temperate forest was reflected by higher C and N contents compared to the semiarid forest. Regression lines between δ13C and -[ln(%C)] showed a stronger isotopic fractionation from top- to subsoil at the semiarid open forest, indicating a faster SOM turnover compared to the humid temperate forest. This is the result of more favorable soil conditions (esp. temperature and smaller C/N ratios) in the semiarid forest. Depth trends of δ15N values indicated N limitation in both soils, though the limitation at the temperate site was stronger. The activity of enzymes degrading cellulose and hemicellulose increased with C content. Activity of enzymes involved in C, N and P cycles decreased from top- to subsoil and

  1. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    Science.gov (United States)

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  2. Impact of chlortetracycline and sulfapyridine antibiotics on soil enzyme activities

    Science.gov (United States)

    Molaei, Ali; Lakzian, Amir; Datta, Rahul; Haghnia, Gholamhosain; Astaraei, Alireza; Rasouli-Sadaghiani, MirHassan; Ceccherini, Maria T.

    2017-10-01

    Pharmaceutical antibiotics are frequently used in the livestock and poultry industries to control infectious diseases. Due to the lack of proper guidance for use, the majority of administrated antibiotics and their metabolites are excreted to the soil environment through urine and feces. In the present study, we used chlortetracycline and sulfapyridine antibiotics to screen out their effects on dehydrogenase, alkaline phosphatase and urease activity. Factorial experiments were conducted with different concentrations of antibiotic (0, 10, 25 and 100 mg kg-1 of soil) mixed with soil samples, and the enzyme activity was measured at intervals of 1, 4 and 21 days. The results show that the chlortetracycline and sulfapyridine antibiotics negatively affect the dehydrogenase activity, but the effect of sulfapyridine decreases with time of incubation. Indeed, sulfapyridine antibiotic significantly affect the alkaline phosphatase activity for the entire three-time interval, while chlortetracycline seems to inhibit its activity within 1 and 4 days of incubation. The effects of chlortetracycline and sulfapyridine antibiotics on urease activity appear similar, as they both significantly affect the urease activity on day 1 of incubation. The present study concludes that chlortetracycline and sulfapyridine antibiotics have harmful effects on soil microbes, with the extent of effects varying with the duration of incubation and the type of antibiotics used.

  3. A modern mode of activation for nucleic acid enzymes.

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    2007-07-01

    Full Text Available Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes, a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

  4. Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja crops

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    Sinar David Granada García

    2014-09-01

    Full Text Available Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.. Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

  5. Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR and concomitant activation of gelatinolytic enzymes.

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    Synnøve Magnussen

    Full Text Available The urokinase plasminogen activator receptor (uPAR is associated with poor prognosis in oral squamous cell carcinoma (OSCC, and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational

  6. Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

    Science.gov (United States)

    Magnussen, Synnøve; Hadler-Olsen, Elin; Latysheva, Nadezhda; Pirila, Emma; Steigen, Sonja E.; Hanes, Robert; Salo, Tuula; Winberg, Jan-Olof; Uhlin-Hansen, Lars; Svineng, Gunbjørg

    2014-01-01

    Background The urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells’ expression level of uPAR affected the activity of gelatinolytic enzymes. Methods The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography. Results We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes. Conclusions Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the

  7. Changes of enzyme activities in lens after glaucoma trabecular resection

    Directory of Open Access Journals (Sweden)

    Jian-Ping Wang

    2013-08-01

    Full Text Available AIM: To observe the change of lens antioxidant enzyme activity after glaucoma trabecular resection. METHODS: Thirty-two eyes of sixteen New-Zealand rabbits(2.2-2.4kgwere divided into two groups. The left eyes of rabbits underwent standard glaucoma trabecular resection were treatment group, and the normal right eyes served as controls. Transparency of lenses was monitored by a slit-lamp biomicroscopy before and after glaucoma trabecular resection. The morphology of lens cells was observed under the light microscope.The activities of Na+-K+-ATPase,catalase(CAT, glutathion peroxidase(GSH-px, glutathione reductase(GR, superoxide dismutase(SODand content of malondialdehyde(MDAin lenses were detected six months after trabecular resection. RESULTS: Lenses were clear in both treatment group and normal control group during the six months after operation. The morphology and structure of lens cells were normal under the light microscope in both operation group and normal group. The activity of lens cells antioxidant enzyme activity were significantly decreased in operation group compared with control group, Na+-K+-ATPase declined by 20.97%, CAT declined by 16.36%, SOD declined by 4.46%, GR declined by 4.85%, GSH-px declined by 10.02%, and MDA increased by 16.31%. CONCLUSION: Glaucoma trabecular resection can induce the change of Na+-K+-ATPase, CAT, GSH-px, GR, SOD and MDA in lens of rabbit. Glaucoma filtration surgery for the occurrence of cataract development mechanism has important guiding significance.

  8. Puromycin-sensitive aminopeptidase: an antiviral prodrug activating enzyme.

    Science.gov (United States)

    Tehler, Ulrika; Nelson, Cara H; Peterson, Larryn W; Provoda, Chester J; Hilfinger, John M; Lee, Kyung-Dall; McKenna, Charles E; Amidon, Gordon L

    2010-03-01

    Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al., 2008. Molecular Pharmaceutics 5, 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k(cat) for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k(cat) for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.

  9. Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiaojun; Minasov, George; Shoichet, Brian K. (NWU)

    2010-03-08

    Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the {beta}-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 {yields} Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

  10. Characterization of Co-Cultivation of Cyanobacteria on Growth, Productions of Polysaccharides and Extracellular Proteins, Nitrogenase Activity, and Photosynthetic Activity.

    Science.gov (United States)

    Xue, Chuizhao; Wang, Libo; Wu, Tong; Zhang, Shiping; Tang, Tao; Wang, Liang; Zhao, Quanyu; Sun, Yuhan

    2017-01-01

    Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N 2 -fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.

  11. Adsorption of Trametes versicolor laccase to soil iron and aluminum minerals: enzyme activity, kinetics and stability studies.

    Science.gov (United States)

    Wu, Yue; Jiang, Ying; Jiao, Jiaguo; Liu, Manqiang; Hu, Feng; Griffiths, Bryan S; Li, Huixin

    2014-02-01

    Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26-64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  12. Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

    Science.gov (United States)

    Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

    2014-04-01

    Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

  13. Escherichia coli-derived and Staphylococcus aureus-derived extracellular vesicles induce MUC5AC expression via extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

    Science.gov (United States)

    Bae, Chang Hoon; Choi, Yoon Seok; Song, Si-Youn; Kim, Yoon-Keun; Kim, Yong-Dae

    2017-01-01

    Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) release extracellular vesicles (EVs). E. coli-derived and S. aureus-derived EVs are associated with neutrophilic respiratory inflammation. In neutrophilic respiratory inflammation of human, expression of mucin is increased in airway epithelial cells and is associated with increased morbidity and mortality of the affected patients. However, no study on the effects of EVs on expression of mucin genes has been reported in airway epithelial cells. Therefore, this study was conducted in order to examine the effects and the brief signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects and signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression were examined using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). E. coli-derived and S. aureus-derived EVs induced MUC5AC expression. E. coli-derived and S. aureus-derived EVs significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked E. coli-derived and S. aureus-derived EVs induced MUC5AC messenger RNA (mRNA) expression. The results of this study suggest that E. coli-derived and S. aureus-derived EVs induced MUC5AC expression via ERK1/2 and p38 MAPK signaling pathways in human airway epithelial cells. © 2016 ARS-AAOA, LLC.

  14. County-Scale Spatial Distribution of Soil Enzyme Activities and Enzyme Activity Indices in Agricultural Land: Implications for Soil Quality Assessment

    Directory of Open Access Journals (Sweden)

    Xiangping Tan

    2014-01-01

    Full Text Available Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2 scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI and the geometric mean of enzyme activities (GME. At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

  15. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Joshua Kellogg

    2014-10-01

    Full Text Available Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively. The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia.

  16. Endothelial cells suppress monocyte activation through secretion of extracellular vesicles containing antiinflammatory microRNAs.

    Science.gov (United States)

    Njock, Makon-Sébastien; Cheng, Henry S; Dang, Lan T; Nazari-Jahantigh, Maliheh; Lau, Andrew C; Boudreau, Emilie; Roufaiel, Mark; Cybulsky, Myron I; Schober, Andreas; Fish, Jason E

    2015-05-14

    The blood contains high concentrations of circulating extracellular vesicles (EVs), and their levels and contents are altered in several disease states, including cardiovascular disease. However, the function of circulating EVs, especially the microRNAs (miRNAs) that they contain, are poorly understood. We sought to determine the effect of secreted vesicles produced by quiescent endothelial cells (ECs) on monocyte inflammatory responses and to assess whether transfer of microRNAs occurs between these cells. We observed that monocytic cells cocultured (but not in contact) with ECs were refractory to inflammatory activation. Further characterization revealed that endothelium-derived EVs (EC-EVs) suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses. EVs isolated from mouse plasma also suppressed monocyte activation. Importantly, injection of EC-EVs in vivo repressed monocyte/macrophage activation, confirming our in vitro findings. We found that several antiinflammatory microRNAs were elevated in EC-EV-treated monocytes. In particular, miR-10a was transferred to monocytic cells from EC-EVs and could repress inflammatory signaling through the targeting of several components of the NF-κB pathway, including IRAK4. Our findings reveal that ECs secrete EVs that can modulate monocyte activation and suggest that altered EV secretion and/or microRNA content may affect vascular inflammation in the setting of cardiovascular disease. © 2015 by The American Society of Hematology.

  17. The effect of active and passive intravenous cocaine administration on the extracellular signal-regulated kinase (ERK) activity in the rat brain.

    Science.gov (United States)

    Miszkiel, Joanna; Detka, Jan; Cholewa, Joanna; Frankowska, Małgorzata; Nowak, Ewa; Budziszewska, Bogusława; Przegaliński, Edmund; Filip, Małgorzata

    2014-08-01

    According to a current hypothesis of learning processes, recent papers pointed out to an important role of the extracellular signal-regulated kinase (ERK), in drug addiction. We employed the Western blotting techniques to examine the ERK activity immediately after cocaine iv self-administration and in different drug-free withdrawal periods in rats. To distinguish motivational vs. pharmacological effects of the psychostimulant intake, a "yoked" procedure was used. Animals were decapitated after 14 daily cocaine self-administration sessions or on the 1st, 3rd or 10th extinction days. At each time point the activity of the ERK was assessed in several brain structures, including the prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens. Passive, repeated iv cocaine administration resulted in a 45% increase in ERK phosphorylation in the hippocampus while cocaine self-administration did not change brain ERK activity. On the 1st day of extinction, the activity of the ERK in the prefrontal cortex was decreased in rats with a history of cocaine chronic intake: by 66% for "active" cocaine group and by 35% for "yoked" cocaine group. On the 3rd day the reduction in the ERK activity (25-34%) was observed in the hippocampus for both cocaine-treated groups, and also in the nucleus accumbens for "yoked" cocaine group (40%). On the 10th day of extinction there was no significant alteration in ERK activity in any group of rats. Our findings suggest that cortical ERK is involved in cocaine seeking behavior in rats. They also indicate the time and regional adaptations in this enzyme activity after cocaine withdrawal. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. Milk clotting and proteolytic activity of enzyme preparation from ...

    African Journals Online (AJOL)

    Some microorganisms have the ability to produce enzymes that could clot milk and used as a substitute for calf rennet. Strains of lactic acid bacteria (LAB) could produce proteolytic enzymes that may have the potential to be used as a source of milk clotting enzyme (MCE). In the present study, LAB isolated from shrimp paste ...

  19. Effect of thymol and carvacrol feed supplementation on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities, and immune response in broiler chickens

    NARCIS (Netherlands)

    Hashemipour, H.; Kermanshahi, H.; Golian, A.; Veldkamp, T.

    2013-01-01

    This trial was conducted to evaluate the effects of dietary supplementation of phytogenic product containing an equal mixture of thymol and carvacrol at 4 levels (0, 60, 100, and 200 mg/kg of diet) on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities,

  20. A study of enzyme and protein microencapsulation--some factors affecting the low apparent enzymic activity yields.

    Science.gov (United States)

    Wood, D A; Whateley, T L

    1982-09-01

    Microencapsulation of aqueous solutions of enzymes, alpha-chymotrypsin (EC 3.4.21.1) and histidase (EC 4.3.1.3), with semipermeable polyamide membranes resulted in a loss of enzymic activity. The low yields (less than 40%) found with both enzymes were typical of others reported in the literature. The activity of broken histidase-containing microcapsules was greater than that of the microcapsules before breaking, and this was interpreted as being due to a simple diffusional restriction on the substrate and product. The maximum of the apparent pH-activity curve of alpha-chymotrypsin was found to be shifted one unit to more alkaline pH when the enzyme was encapsulated. This phenomenon was explained in terms of the hydrogen ion concentration in the microenvironment surrounding the enzyme being different from that in the bulk solution. Microencapsulation of aqueous solutions of enzymes is accomplished by in situ polymerization reactions at the interface of a water-in-oil emulsion. 125I-labelled proteins (albumin and fibrinogen) were encapsulated under similar conditions to determine the efficiency of the microencapsulation process. About one third of these proteins was lost during the overall preparation procedure and a further fraction was attached to the membranes of the microcapsules.

  1. Effects of Fertilization on Tomato Growth and Soil Enzyme Activity

    Science.gov (United States)

    Mu, Zhen; Hu, Xue-Feng; Cheng, Chang; Luo, Zhi-qing

    2015-04-01

    To study the effects of different fertilizer applications on soil enzyme activity, tomato plant growth and tomato yield and quality, a field experiment on tomato cultivation was carried out in the suburb of Shanghai. Three fertilizer treatments, chemical fertilizer (CF) (N, 260 g/kg; P, 25.71g/kg; K, 83.00g/kg), rapeseed cake manure (CM) (N, 37.4 g/kg; P, 9.0 g/kg; K, 8.46 g/kg), crop-leaf fermenting manure (FM) (N, 23.67 g/kg; P, 6.39 g/kg; K 44.32 g/kg), and a control without using any fertilizers (CK), were designed. The total amounts of fertilizer application to each plot for the CF, CM, FM and CK were 0.6 kg, 1.35 kg, 3.75 kg and 0 kg, respectively, 50% of which were applied as base fertilizer, and another 50% were applied after the first fruit picking as top dressing. Each experimental plot was 9 m2 (1 m × 9 m) in area. Each treatment was replicated for three times. No any pesticides and herbicides were applied during the entire period of tomato growth to prevent their disturbance to soil microbial activities. Soil enzyme activities at each plot were constantly tested during the growing period; the tomato fruit quality was also constantly analyzed and the tomato yield was calculated after the final harvesting. The results were as follows: (1) Urease activity in the soils treated with the CF, CM and FM increased quickly after applying base fertilizer. That with the CF reached the highest level. Sucrase activity was inhibited by the CF and CM to some extent, which was 32.4% and 11.2% lower than that with the CK, respectively; while that with the FM was 15.7% higher than that with the CK. Likewise, catalase activity with the CF increased by 12.3% - 28.6%; that with the CM increased by 87.8% - 95.1%; that with the FM increased by 86.4% - 93.0%. Phosphatase activity with the CF increased rapidly and reached a maximum 44 days after base fertilizer application, and then declined quickly. In comparison, that with the CM and FM increased slowly and reached a maximum

  2. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    Science.gov (United States)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  3. Aberrant activity of the DNA repair enzyme AlkB.

    Science.gov (United States)

    Henshaw, Timothy F; Feig, Michael; Hausinger, Robert P

    2004-05-01

    Escherichia coli AlkB is a DNA/RNA repair enzyme containing a mononuclear Fe(II) site that couples the oxidative decomposition of alpha-ketoglutarate (alphaKG) to the hydroxylation of 1-methyladenine or 3-methylcytosine lesions in DNA or RNA, resulting in release of formaldehyde and restoration of the normal bases. In the presence of Fe(II), alphaKG, and oxygen, but the absence of methylated DNA, AlkB was found to catalyze an aberrant reaction that generates a blue chromophore. The color is proposed to derive from Fe(III) coordinated by a hydroxytryptophan at position 178 as revealed by mass spectrometric analysis. Protein structural modeling confirms that Trp 178 is reasonably positioned to react with the Fe(IV)-oxo intermediate proposed to form at the active site.

  4. The effect of aluminium on enzyme activities in two wheat cultivars

    African Journals Online (AJOL)

    Administrator

    2011-04-25

    Apr 25, 2011 ... In order to study the effect of different aluminum (Al) concentrations on the enzyme activities of wheat seedlings and ... activity of different enzymes such as ascorbate peroxidase (APX), catalase (CAT), glutathione reductase. (GR) and ...... antioxidant enzymes by salt stress in the rice (Oryza sativa L.). Plant.

  5. Effect of eosinophils activated with Alternaria on the production of extracellular matrix from nasal fibroblasts.

    Science.gov (United States)

    Shin, Seung-Heon; Ye, Mi-Kyung; Choi, Sung-Yong; Kim, Yee-Hyuk

    2016-06-01

    Eosinophils and fibroblasts are known to play major roles in the pathogenesis of nasal polyps. Fungi are commonly found in nasal secretion and are associated with airway inflammation. To investigate whether activated eosinophils by airborne fungi can influence the production of extracellular matrix (ECM) from nasal fibroblasts. Inferior turbinate and nasal polyp fibroblasts were stimulated with Alternaria or Aspergillus, respectively, for 24 hours and ECM messenger RNA (mRNA) and protein expressions were measured. Eosinophils isolated from healthy volunteers were stimulated with Alternaria or Aspergillus for 4 hours then superoxide, eosinophil peroxidase, and transforming growth factor β1 were measured. Then activated eosinophils were cocultured with nasal fibroblasts for 24 hours, and ECM mRNA expressions were measured. Alternaria strongly enhanced ECM mRNA expression and protein production from nasal fibroblasts. Alternaria also induced the production of superoxide, eosinophil peroxidase, and transforming growth factor β1 from eosinophils, and activated eosinophils enhanced ECM mRNA expression when they were cocultured without the Transwell insert system. Eosinophils activated with Alternaria enhanced ECM mRNA expression from nasal polyp fibroblasts. Alternaria plays an important role in tissue fibrosis in the pathogenesis of nasal polyps by directly or indirectly influencing the production of ECM from nasal fibroblasts. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains...... on the receptor already lacking the second disulfide bridge and already displaying a high Zn (2+) potency. We conclude that the second disulfide bridge, which according to the beta2-adrenergic structure will form a covalent link across the entrance to the main ligand binding pocket, serves to dampen GPR39...... activation. We suggest that formation of extra disulfide bridges may be an important general mechanism for regulating the activity of 7TM receptors....

  7. Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments.

    Science.gov (United States)

    Zhang, Xiaoxu; Gao, Yanyun; Yin, Ying; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2017-02-07

    Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.

  8. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs.

    Directory of Open Access Journals (Sweden)

    Yujin Tang

    Full Text Available Vacuolar processing enzymes (VPEs have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD, which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177, Cys (219] and substrate binding pocket [Arg (112, Arg (389, Ser (395], except that Ser (395 in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF, close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

  9. Temporal variations in microbial biomass C and cellulolytic enzyme activity in arable soils: effects of organic matter input

    DEFF Research Database (Denmark)

    Debosz, K.; Rasmussen, Peter Have; Pedersen, A. R.

    1999-01-01

    growth, i.e. by factors common to both systems but not differences in organic matter input. Pronounced and constant increases in beta-glucosidase activity (40%) and endocellulase activity (30%) in high-OM were detected across all sampling periods. The increases in microbial biomass C concentration......Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic complex were investigated in a field experiment after eight years of cultivation with either low organic matter input (low-OM) or high organic matter input (high......-OM). The cultivation systems differed in whether their source of fertiliser was mainly mineral or organic, in whether a winter cover crop was grown, and whether straw was mulched or removed. Sampling occurred at approximately monthly intervals, over a period of two years. Distinct temporal variations in microbial...

  10. Temporal variations in microbial biomass C and cellulolytic enzyme activity in arable soils: effects of organic matter input

    DEFF Research Database (Denmark)

    Debosz, K.; Rasmussen, Peter Have; Pedersen, A. R.

    1999-01-01

    Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic complex were investigated in a field experiment after eight years of cultivation with either low organic matter input (low-OM) or high organic matter input (high......-OM). The cultivation systems differed in whether their source of fertiliser was mainly mineral or organic, in whether a winter cover crop was grown, and whether straw was mulched or removed. Sampling occurred at approximately monthly intervals, over a period of two years. Distinct temporal variations in microbial...... growth, i.e. by factors common to both systems but not differences in organic matter input. Pronounced and constant increases in beta-glucosidase activity (40%) and endocellulase activity (30%) in high-OM were detected across all sampling periods. The increases in microbial biomass C concentration...

  11. A cytochemical method for measuring enzyme activity in individual preovulatory mouse oocytes

    NARCIS (Netherlands)

    de Schepper, G. G.; van Noorden, C. J.; Koperdraad, F.

    1985-01-01

    The activities of 6 enzymes involved in carbohydrate metabolism were determined quantitatively in preovulatory oocytes by cytochemical means per individual cell as well as biochemically in cell homogenates. Oocytes were incorporated in a polyacrylamide matrix for appropriate enzyme cytochemical

  12. Cell survival, activation and apoptosis of hepatic stellate cells: modulation by extracellular matrix proteins.

    Science.gov (United States)

    Priya, Sulochana; Sudhakaran, Perumana R

    2008-12-01

    Cytokines and growth factors released by various hepatic cells exert both paracrine and autocrine effects on hepatic stellate cell (HSC) activation during liver injury. The aim of the present study was to examine whether the surrounding extracellular matrix (ECM) influences the activation, transdifferentiation and survival of HSCs. An in vitro model system of isolated HSCs maintained in culture on different matrix protein substrata was employed. The rate of loss of HSC-specific retinol uptake activity and gain of myofibroblast-like activity such as (35)[S] proteoglycan synthesis varied in cells maintained on different matrix proteins and was in the order collagen I > collagen IV >/= laminin. (3)[H]-thymidine incorporation by HSCs maintained on different matrix proteins varied and was in the order collagen I > collagen IV > laminin. MTT assay revealed that the growth inhibition in response to curcumin was significantly low in cells maintained on collagen I. Apoptotic marker activities such as DNA fragmentation, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, annexin staining and caspase-3 activities showed that cells maintained on collagen I showed minimal apoptosis than those maintained on collagen IV, laminin and polylysine, showing the influence of ECM on HSC apoptosis. Experiments using blocking antibodies showed that the collagen I effect was mediated through alpha(2)beta(1) integrin. These results indicate that ECM influences activation, transdifferentiation and survival of HSCs, and suggest that apart from diffusible factors, the surrounding ECM also influences HSC behavior critical in both the progression of the fibrosis and the restitution of the liver during recovery after hepatic injury.

  13. Antibodies to the extracellular pore loop of TRPM8 act as antagonists of channel activation.

    Directory of Open Access Journals (Sweden)

    Silke Miller

    Full Text Available The mammalian transient receptor potential melastatin channel 8 (TRPM8 is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced 45Ca2+ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies.

  14. Intracellular modulation, extracellular disposal and serum increase of MiR-150 mark lymphocyte activation.

    Directory of Open Access Journals (Sweden)

    Paola de Candia

    Full Text Available Activated lymphocytes release nano-sized vesicles (exosomes containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

  15. Regulation of extracellular calcium in the hippocampus in vivo during epileptiform activity – role of astrocytes

    Science.gov (United States)

    Stringer, Janet L.; Mukherjee, Kakali; Xiang, Ting; Xu, Kaiping

    2007-01-01

    Astrocytes have been suggested to regulate the extracellular calcium concentration ([Ca2+]o), but this has not been thoroughly investigated. Adult, male Sprague-Dawley rats were used to record changes in [Ca2+]o in the hippocampus during epileptiform activity. Maximal decreases in [Ca2+]o in CA1 were measured in the pyramidal cell layer during 20 Hz, 20 sec stimulus trains to the contralateral CA3 region. Maximal decreases in [Ca2+]o in the dentate gyrus were measured when maximal dentate activation had appeared - irrespective of the location, frequency or duration of the stimulation. Maximal decreases were 36% greater in the dentate gyrus than in CA1. During prolonged discharges, [Ca2+]o recovered partially towards the baseline in both hippocampal regions. To investigate the role of astrocytes, local injections of fluorocitrate (FC), a metabolic toxin selectively taken up by astrocytes, were used. FC (0.1, 0.25 or 0.5 mM FC), but not vehicle (2 μl), caused a small, but significant decrease in the maximal changes in CA1, but an increase in the dentate gyrus. The results suggest that maximal decreases in [Ca2+]o occur in the hippocampus in response to burst firing of neurons and that astrocytes play a minimal role in the regulation of [Ca2+]o during epileptiform activity. PMID:17434291

  16. Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

    Directory of Open Access Journals (Sweden)

    Natalia Fullana

    2017-08-01

    Full Text Available Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10 from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20 and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.

  17. Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

    DEFF Research Database (Denmark)

    Petersen, R H; Knudsen, T; Johansen, Torben

    1991-01-01

    The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat...... and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation...... of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium....

  18. Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

    Science.gov (United States)

    Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

    2015-01-01

    Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

  19. Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.

    Science.gov (United States)

    Ermert, Susanne; Marx, Andreas; Hacker, Stephan M

    2017-04-01

    Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

  20. Enzyme catalysis: C-H activation is a Reiske business

    Science.gov (United States)

    Bruner, Steven D.

    2011-05-01

    Enzymes that selectively oxidize unactivated C-H bonds are capable of constructing complex molecules with high efficiency. A new member of this enzyme family is RedG, a Reiske-type oxygenase that catalyses chemically challenging cyclizations in the biosynthesis of prodiginine natural products.

  1. Physicochemical Properties and Enzymes Activity Studies in a ...

    African Journals Online (AJOL)

    Soil Physicochemical properties and enzyme concentration were evaluated in soil from a refined-oil contaminated community in Isiukwuato, Abia State three years after the spill. The soil enzymes examined were urease, lipase, oxidase, alkaline and acid phosphatases. Results show a significant (P< 0.05) decrease in the ...

  2. Enzyme activities and histopathology of selected tissues in rats ...

    African Journals Online (AJOL)

    The effect of chronic administration of potassium bromate (KBrO3), a flour improver, on some 'marker' enzymes of rat cellular system was investigated. The levels of these enzymes were measured progressively in the kidney, liver and small intestine, 24h after days 1, 3, 5, 10, 15 and 20 following the administration of ...

  3. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  4. Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Pilgaard, Bo; Lezyk, Mateusz Jakub

    2017-01-01

    and the lytic polysaccharide monooxygenase families. This approach notonly assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy. Results: We identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition......Background: Carbohydrate-active enzymes are found in all organisms and participate in key biological processes.These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis...... for prediction of enzyme function. A fastand reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interestas demonstrated for the glycosyl hydrolase...

  5. Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity

    DEFF Research Database (Denmark)

    Albrechtsen, Reidar; Hansen, Dorte Stautz; Sanjay, Archana

    2011-01-01

    Invadopodia are dynamic actin structures at the cell surface that degrade extracellular matrix and act as sites of signal transduction. The biogenesis of invadopodia, including the mechanisms regulating their formation, composition, and turnover is not entirely understood. Here, we demonstrate...

  6. Continuous enzyme-coupled assay for microbial transglutaminase activity.

    Science.gov (United States)

    Oteng-Pabi, Samuel K; Keillor, Jeffrey W

    2013-10-15

    Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-ε-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG's promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K(M)=3 mM) than the commonly used Cbz-Gln-Gly (K(M)=58 mM), attesting to its potential for application in biocatalysis and bioconjugation. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Investigation of the Extracellular Matrix Effect for the QCM/CCD Cell Activity Monitoring System.

    Science.gov (United States)

    Kang, Hyen-Wook; Otani, Naoya; Hiroshi, Muramatsu; Chang, Sang-Mok; Kim, Jong Min

    2018-08-01

    A real-time quartz crystal microbalance (QCM) cell activity monitoring system coupled with micro CCD cameras was developed to investigate the cultured cell activity, which could measure the viscoelastic characteristics of the cell with the QCM and observe the cell morphology changes with CCD camera simultaneously. Both the viscoelastic characteristics and the shape of the cultured cell are important factors to estimate the cell activity and the cell adhesion. The extracellular matrix (ECM) on the surface of the QCM is essential to culture the cell stably in the QCM monitoring system. To find the ECM optimization condition, the adhesive strength of cultured cells on the ECM modified glass surface was measured by using rotating water stream and CCD camera. After culturing HepG2 cells for 24 hours on the ECM modified glass plates, the glass plates were dipped in the PBS solution and rotated with 1,000, 1,300, and 1,500 rpm for 30 seconds. The adhesiveness of ECMs was investigated by calculating the remained cells after rotating. Four types of ECM, such as amino group, carboxyl group, collagen monomer, and collagen polymer, were used and tested. The current paper improves the sensing system of previous report so that measurements of four ECMs can be simultaneously conducted under the same conditions in order to enhance reliability. A collagen polymer exposed ECM was the most stable on an adhesiveness point of view, but not suitable for the QCM cell activity monitoring due to the decrease of the QCM sensitivity. The sensitivity of the QCM cell activity monitoring system using collagen monomer as ECM is about 2.6 times better than that using collagen polymer. A collagen monomer exposed ECM was more stable than amino group and carboxyl group exposed ECMs based on an adhesiveness point of view. Therefore, a collagen monomer exposed ECM was the most stable and suitable for the QCM cell activity monitoring system among the four ECMs. The changes of the resonance frequency

  8. Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

    Directory of Open Access Journals (Sweden)

    Abdullah A Gharamah

    2014-01-01

    Full Text Available Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2, sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin. Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss.

  9. Escherichia coli and Candida albicans induced macrophage extracellular trap-like structures with limited microbicidal activity.

    Science.gov (United States)

    Liu, Pan; Wu, Xiuping; Liao, Chengshui; Liu, Xiaolei; Du, Jing; Shi, Haining; Wang, Xuelin; Bai, Xue; Peng, Peng; Yu, Lu; Wang, Feng; Zhao, Ying; Liu, Mingyuan

    2014-01-01

    The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to Escherichia coli and Candida albicans challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped E. coli but not C. albicans. Fluorescence and scanning electron microscopy analyses revealed that, in vitro, both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.

  10. Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

    Science.gov (United States)

    Wilhelm, Konrad; Ganesan, Jayanthi; Müller, Tobias; Dürr, Christoph; Grimm, Melanie; Beilhack, Andreas; Krempl, Christine D; Sorichter, Stephan; Gerlach, Ulrike V; Jüttner, Eva; Zerweck, Alf; Gärtner, Frank; Pellegatti, Patrizia; Di Virgilio, Francesco; Ferrari, Davide; Kambham, Neeraja; Fisch, Paul; Finke, Jürgen; Idzko, Marco; Zeiser, Robert

    2010-12-01

    Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

  11. Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry.

    Science.gov (United States)

    Holnthoner, Wolfgang; Bonstingl, Cornelia; Hromada, Carina; Muehleder, Severin; Zipperle, Johannes; Stojkovic, Stefan; Redl, Heinz; Wojta, Johann; Schöchl, Herbert; Grillari, Johannes; Weilner, Sylvia; Schlimp, Christoph J

    2017-06-16

    Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.

  12. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  13. Extracellular microvesicle production by human eosinophils activated by “inflammatory” stimuli

    Directory of Open Access Journals (Sweden)

    Praveen Akuthota

    2016-10-01

    Full Text Available A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs, very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1 and tumor necrosis factor alpha (TNF-α. EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVsoutwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells.TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20-1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune

  14. Multi-tasking Sulf1/Sulf2 enzymes do not only facilitate extracellular cell signalling but also participate in cell cycle related nuclear events.

    Science.gov (United States)

    Krishnakumar, Kavithanjali; Chakravorty, Ishani; Foy, Wendy; Allen, Steve; Justo, Tiago; Mukherjee, Abir; Dhoot, Gurtej K

    2018-03-01

    This study demonstrates highly dynamic spatial and temporal pattern of SULF1/SULF2 expression in a number of neuronal cell types growing in normal culture medium that included their transient nuclear mobilisation. Their nuclear translocation became particularly apparent during cell proliferation as both SULF1/SULF2 demonstrated not only cell membrane associated expression, their known site of function but also transient nuclear mobilisation during nuclear cell division. Nuclear localisation was apparent not only by immunocytochemical staining but also confirmed by immunoblotting staining of isolated nuclear fractions of C6, U87 and N2A cells. Immunocytochemical analysis demonstrated rapid nuclear exit of both SULF1/SULF2 following cell division that was slightly delayed but not blocked in a fraction of the polyploid cells observed in C6 cells. The overexpression of both Sulf1 and Sulf2 genes in C6 and U87 cells markedly promoted in vitro growth of these cells accompanied by nuclear mobilisation while inhibition of both these genes inhibited cell proliferation with little or no nuclear SULF1/SULF2 mobilisation. SULF1/SULF2 activity in these cells thus demonstrated a clear co-ordination of extracellular cell signalling with nuclear events related to cell proliferation. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

  15. Involvement of neuronal and glial activities in control of the extracellular d-serine concentrations by the AMPA glutamate receptor in the mouse medial prefrontal cortex.

    Science.gov (United States)

    Ishiwata, Sayuri; Umino, Asami; Nishikawa, Toru

    2017-09-28

    It has been well accepted that d-serine may be an exclusive endogenous coagonist for the N-methyl-d-aspartate (NMDA)-type glutamate receptor in mammalian forebrain regions. We have recently found by using an in vivo dialysis method that an intra-medial prefrontal cortex infusion of S-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (S-AMPA), a selective AMPA-type glutamate receptor agonist, causes a reduction in the extracellular levels of d-serine in a calcium-permeable AMPA receptor antagonist-sensitive manner. The inhibitory influence by the AMPA receptor on the extracellular d-serine, however, contradicts the data obtained from in vitro experiments that the AMPA receptor stimulation leads to facilitation of the d-serine liberation. This discrepancy appears to be due to the different cell setups between the in vivo and in vitro preparations. From the viewpoints of the previous reports indicating (1) the neuronal presence of d-serine synthesizing enzyme, serine racemase, and d-serine-like immunoreactivity and (2) the same high tissue concentrations of d-serine in the glia-enriched white matter and in the neuron-enriched gray matter of the mammalian neocortex, we have now investigated in the mouse medial prefrontal cortex, the effects of attenuation of neuronal and glial activities, by tetrodotoxin or fluorocitrate, respectively, on the S-AMPA-induced downregulation of the extracellular d-serine contents. In vivo dialysis studies revealed that a local infusion of tetrodotoxin or fluorocitrate eliminated the ability of S-AMPA given intra-cortically to cause a significant decrease in the dialysate concentrations of d-serine without affecting the elevating effects of S-AMPA on those of glycine, another intrinsic coagonist for the NMDA receptor. These findings suggest that the control by the AMPA receptor of the extracellular d-serine levels could be modulated by the neuronal and glial activities in the prefrontal cortex. It cannot be excluded that

  16. Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Holm, Rikke; Vilsen, Bente

    2016-01-01

    , in addition, Na+/K+-ATPase-mediated K+ clearance could be governed by astrocytic [Na+]i. During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+-coupled glutamate transporters, thereby elevating [Na+]i. It thus remains unresolved whether the different Na......+/K+-ATPase isoforms are controlled by [K+]o or [Na+]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+]o transients with ion-sensitive microelectrodes revealed reduced Na+/K+-ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular...... isoforms than the β2 isoform. In summary, enhanced astrocytic Na+/K+-ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+/K+-ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+]i transients associated with activity...

  17. Chaperone-like activities of α-synuclein: α-Synuclein assists enzyme activities of esterases

    International Nuclear Information System (INIS)

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo; Doohun Kim, T.

    2006-01-01

    α-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of α-synuclein has not yet been known. Here we have shown that α-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of α-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with α-synuclein. Our results indicate that α-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo

  18. Reveal the response of enzyme activities to heavy metals through in situ zymography.

    Science.gov (United States)

    Duan, Chengjiao; Fang, Linchuan; Yang, Congli; Chen, Weibin; Cui, Yongxing; Li, Shiqing

    2018-07-30

    Enzymes in the soil are vital for assessing heavy metal soil pollution. Although the presence of heavy metals is thought to change the soil enzyme system, the distribution of enzyme activities in heavy metal polluted-soil is still unknown. For the first time, using soil zymography, we analyzed the distribution of enzyme activities of alfalfa rhizosphere and soil surface in the metal-contaminated soil. The results showed that the growth of alfalfa was significantly inhibited, and an impact that was most pronounced in seedling biomass and chlorophyll content. Catalase activity (CAT) in alfalfa decreased with increasing heavy metal concentrations, while malondialdehyde (MDA) content continually increased. The distribution of enzyme activities showed that both phosphatase and β-glucosidase activities were associated with the roots and were rarely distributed throughout the soil. In addition, the total hotspot areas of enzyme activities were the highest in extremely heavy pollution soil. The hotspot areas of phosphatase were 3.4%, 1.5% and 7.1% under none, moderate and extremely heavy pollution treatment, respectively, but increased from 0.1% to 0.9% for β-glucosidase with the increasing pollution levels. Compared with the traditional method of enzyme activities, zymography can directly and accurately reflect the distribution and extent of enzyme activity in heavy metals polluted soil. The results provide an efficient research method for exploring the interaction between enzyme activities and plant rhizosphere. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Visualization of Enzyme Activities in Earthworm Biopores by In Situ Soil Zymography.

    Science.gov (United States)

    Razavi, Bahar S; Hoang, Duyen; Kuzyakov, Yakov

    2017-01-01

    Earthworms produce biopores with strongly increased microbial and enzyme activities and consequently they form microbial hotspots in soil. In extremely dynamic microhabitats and hotspots such as earthworm biopores, the in situ enzyme activities are a footprint of process rates and complex biotic interactions. The effect of earthworms on enzyme activities inside biopores, relative to earthworm-free soil, can be visualized by in situ soil zymography. Here, we describe the details of the approach and discuss its advantages and limitations. Direct zymography provides high spatial resolution for quantitative images of enzyme activities in biopores.

  20. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    Science.gov (United States)

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  1. Descriptive and predictive assessment of enzyme activity and enzyme related processes in biorefinery using IR spectroscopy and chemometrics

    DEFF Research Database (Denmark)

    Baum, Andreas

    Enzyme technology provides key strategies to green chemistry as many processes have undergone re-design to serve increasing demands towards being sustainable. While the population is rapidly increasing on our planet it is leading to accumulative problems in terms of production of waste, depletion...... industries. However, as biorefinery concepts are implemented in many industrial processes an increasing demand for Process Analytical Technology (PAT) evolves to monitor, understand and steer processes optimally. Biomasses can be very diverse and are usually of complex chemical nature. Conventional......-order calibration advantage (reference Theory of Analytical chemistry). As PAPER 3 illustrates the method is universally applicable without the need of any external standards and was exemplified by performing quantitative enzyme activity determinations for glucose oxidase, pectin lyase and a cellolytic enzyme blend...

  2. Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production, optimization and enzymatic characterization.

    Science.gov (United States)

    Gastelum-Arellanez, Argel; Paredes-López, Octavio; Olalde-Portugal, Víctor

    2014-11-01

    Endoglucanase activity produced by Paenibacillus polymyxa BEb-40 was studied. In submerged culture with minimal medium supplemented with carboxymethylcellulose (CMC), this microorganism produced up to 0.37 U/mL endoglucanase activity with high specific activity (14.3 U/mgtotal protein). Detection of endoglucanase activity through zymography revealed at least 14 isoenzymes with molecular weights between 38 and 220 kDa. This high variety of secreted endoglucanases has not been described previously in Paenibacillus genus. The optimum conditions, determined by response surface methodology, were 48 °C and pH 3.4, which allowed an increase of 33.7 % in the relative endoglucanase activity obtained with respect to the standard conditions. Nevertheless, high levels of hydrolysis of at least 70 % of the maximum activity could be obtained at wide ranges of pH (2-9) and temperature (40-60 °C). Under optimal conditions, high levels of CMC hydrolysis were reached, of about 40 %, after only 12 h of reaction with substrate/total protein ratios between 19 and 76. Kinetic analysis revealed that endoglucanase activity followed a mixed inhibition model (K m = 8.4 mM, K ic = 0.03 mM, K iu = 0.35 mM, V max = 33.3 U/mgtotal protein). These results allow to consider P. polymyxa BEb-40 as a promising microorganism for the production of endoglucanases, with possibilities of application in the breakdown of lignocellulosic biomass. The high specific activity at wide ranges of pH and temperature can allow its use in a wide variety of processes, under both acidic and alkaline conditions, as well as in mesophilic and thermophilic temperatures, further reducing the amount of enzymes used.

  3. Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity

    Science.gov (United States)

    Ouyang, Yingshi; Bayer, Avraham; Chu, Tianjiao; Tyurin, Vladimir A.; Kagan, Valerian E.; Morelli, Adrian E.; Coyne, Carolyn B; Sadovsky, Yoel

    2016-01-01

    Introduction Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. PMID:27780544

  4. Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.

    Science.gov (United States)

    Niu, Ziru; Pang, Ronald T K; Liu, Weimin; Li, Qian; Cheng, Ranran; Yeung, William S B

    2017-01-01

    Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.

  5. Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

    Science.gov (United States)

    Hoang, D. T. T.

    2016-12-01

    Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

  6. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    Science.gov (United States)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  7. Procoagulant activity of extracellular vesicles as a potential biomarker for risk of thrombosis and DIC in patients with acute leukaemia.

    Science.gov (United States)

    Gheldof, Damien; Haguet, Hélène; Dogné, Jean-Michel; Bouvy, Céline; Graux, Carlos; George, Fabienne; Sonet, Anne; Chatelain, Christian; Chatelain, Bernard; Mullier, François

    2017-02-01

    Haemostatic complication is common for patients with hematologic malignancies. Recent studies suggest that the procoagulant activity (PCA) of extracellular vesicles (EV) may play a major role in venous thromboembolism and disseminated intravascular coagulation (DIC) in acute leukaemia. To study the impact of EVs from leukaemic patients on thrombin generation and to assess EV-PCA as a potential biomarker for thrombotic complications in patients with acute leukaemia. Blood samples from a cohort of patients with newly diagnosed acute leukaemia were obtained before treatment (D-0), 3 and 7 days after treatment (D-3 and D-7). Extracellular vesicles were isolated and concentrated by ultracentrifugation. EV-PCA was assessed by thrombin generation assay, and EV-associated tissue factor activity was measured using a commercial bio-immunoassay (Zymuphen MP-TF®). Of the 53 patients, 6 had increased EV-PCA at D-0 and 4 had a thrombotic event. Patients without thrombotic events (n = 47) had no elevated EV-PCA. One patient had increased EVs with procoagulant activity at D-3 and developed a DIC at D-5. This patient had no increased EVs-related tissue factor activity from D-0 to D-7 (2 pg/ml), of these, four had a thrombosis and two had haemorrhages. Procoagulant activity of extracellular vesicles could have a predictive value in excluding the risk of thrombotic events. Our findings also suggest a possible association between thrombotic events and EV-PCA.

  8. Comparison of N-acetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans.

    Science.gov (United States)

    Djordjevic, Natasa; Carrillo, Juan Antonio; Roh, Hyung-Keun; Karlsson, Sara; Ueda, Nobuhisa; Bertilsson, Leif; Aklillu, Eleni

    2012-10-01

    The aim of this study was to compare xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) genotype and phenotype between Swedes (n = 113) and Koreans (n = 150), as well as to investigate the effect of sex, smoking, age, and oral contraceptive (OC) use on enzyme activities, using caffeine as a probe. XO and NAT2 activities were estimated by 1U/(1U+1X) and AFMU/(AFMU+1X+1U) urinary ratios, respectively. Participants were genotyped for 191G>A, 341T>C, 590G>A, and 857G>A NAT2 polymorphisms. There was no significant difference in XO activity between Swedes and Koreans. In Swedes, higher XO activity was observed in women (P < .003). There were significant differences in NAT2 genotype and phenotype between Swedes and Koreans. Koreans display significantly higher frequency of NAT2 fast acetylator genotype (89%), whereas the slow acetylator genotype is predominant (62%) in Swedes (P < .0001). Significantly higher NAT2 activity was observed in Koreans compared to Swedes (P < .0001). Having the same NAT2 fast acetylator genotype, Koreans display higher enzyme activity than Swedes (P < .004). OC use significantly increased NAT2 activity in Swedish women. In conclusion, Koreans display higher NAT2 activity than Swedes regardless of NAT2 genotype. Ethnicity, OC use, and genotype determine NAT2 activity, whereas sex is the only determinant of XO activity.

  9. Effect of Cereal Type and Enzyme Addition on Performance, Pancreatic Enzyme Activity, Intestinal Microflora and Gut Morphology of Broilers

    Directory of Open Access Journals (Sweden)

    Kalantar M

    2016-06-01

    Full Text Available The effects of grain and carbohydrase enzyme supplementation were investigated on digestive physiology of chickens. A total of 625 one-day-old chicks (Ross 308 were randomly assigned to five treatments in a completely randomized design. Treatments included two different types of grains (wheat, and barley with or without a multi-carbohydrase supplement. A corn-based diet was also considered to serve as a control. Feeding barley-based diet with multi-carbohydrase led to higher feed intake (P < 0.01 than those fed corn- and wheat-based diets. Birds fed on barley and wheat diets had lower weight gain despite a higher feed conversion ratio (P < 0.01. Total count and number of different type of bacteria including Gram-negative, E. coli, and Clostridia increased after feeding wheat and barley but the number of Lactobacilli and Bifidobacteria decreased (P < 0.01. Feeding barley and wheat diets reduced villus height in different parts of the small intestine when compared to those fed on a corn diet. However, enzyme supplementation of barley and wheat diets improved weight gain and feed conversion ratio and resulted in reduced number of E. coli and Clostridia and increased number of Lactobacilli and Bifidobacteria, and also restored the negative effects on intestinal villi height (P < 0.01. The activities of pancreatic α-amylase and lipase were (P < 0.01 increased in chickens fed wheat and barley diets when compared to the control fed on a corn diet. Enzyme supplementation reduced the activities of pancreatic α-amylase and lipase (P < 0.01. In conclusion, various dietary non-starch polysaccharides without enzyme supplementation have an adverse effect on digesta viscosity, ileal microflora, villi morphology, and pancreatic enzyme activity.

  10. Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot Spring, Malaysia

    Science.gov (United States)

    Msarah, Marwan; Ibrahim, Izyanti; Aqma, Wan Syaidatul

    2018-04-01

    Thermophilic bacteria have biotechnological importance due to the availability of unique enzymes which are stable in extreme circumstances. The aim of this study includes to isolate thermophilic bacteria from hot spring and screen for important enzyme activities. Water samples from the Dusun Tua Hot Spring were collected and the physiochemical characterisation of water was measured. Eight thermophilic bacteria were isolated and determined to have at least three strong enzyme activity including protease, lipase, amylase, cellulase, pectinase and xylanase. The results showed that HuluC2 displayed all the enzyme activities and can be further studied.

  11. Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Koka, P.

    1984-01-01

    A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

  12. Escherichia coli and Candida albicans induced macrophage extracellular trap-like structures with limited microbicidal activity.

    Directory of Open Access Journals (Sweden)

    Pan Liu

    Full Text Available The formation of extracellular traps (ETs has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17% and peritoneal macrophages (8.5% to 15% form METs-like structures (METs-LS in response to Escherichia coli and Candida albicans challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped E. coli but not C. albicans. Fluorescence and scanning electron microscopy analyses revealed that, in vitro, both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.

  13. Annonacin Exerts Antitumor Activity through Induction of Apoptosis and Extracellular Signal-regulated Kinase Inhibition.

    Science.gov (United States)

    Yap, Chee Voon; Subramaniam, Kavita S; Khor, Sik Wey; Chung, Ivy

    2017-01-01

    Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata , is a potential alternative therapeutic agent to treat EC. To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC 50 values ranging from 4.62 to 4.92 μg/ml. EC cells were shown arrested at G2/M phase after treated with 4 μg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells ( P cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate.

  14. Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

    Directory of Open Access Journals (Sweden)

    Taufik Indarmawan

    2016-04-01

    Full Text Available Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.

  15. Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+ and Calmodulin

    Science.gov (United States)

    Egea, Joaquim; Espinet, Carme; Soler, Rosa M.; Peiró, Sandra; Rocamora, Nativitat; Comella, Joan X.

    2000-01-01

    Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner. PMID:10688641

  16. Enzymes activities involving bacterial cytochromes incorporated in clays

    International Nuclear Information System (INIS)

    Lojou, E.; Giudici-Orticoni, M.Th.; Bianco, P.

    2005-01-01

    With the development of bio electrochemistry, researches appeared on the enzymes immobilization at the surface of electrodes for the realization of bioreactors and bio sensors. One of the main challenges is the development of host matrix able to immobilize the protein material preserving its integrity. In this framework the authors developed graphite electrodes modified by clay films. These electrodes are examined for two enzyme reactions involving proteins of sulfate-reduction bacteria. Then in the framework of the hydrogen biological production and bioreactors for the environmental pollution de-pollution, the electrochemical behavior of the cytochrome c3 in two different clays deposed at the electrode is examined

  17. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation

    Directory of Open Access Journals (Sweden)

    Dora eBrites

    2015-12-01

    Full Text Available Patients with chronic inflammation are often associated with the emergence of depression symptoms, while diagnosed depressed patients show increased levels of circulating cytokines. Further studies revealed the activation of the brain immune cell microglia in depressed patients with a greater magnitude in individuals that committed suicide, indicating a crucial role for neuroinflammation in depression brain pathogenesis. Rapid advances in the understanding of microglial and astrocytic neurobiology were obtained in the past fifteen to twenty years. Indeed, recent data reveal that microglia play an important role in managing neuronal cell death, neurogenesis, and synaptic interactions, besides their involvement in immune-response generating cytokines. The communication between microglia and neurons is essential to synchronize these diverse functions with brain activity. Evidence is accumulating that secreted extracellular vesicles (EVs, comprising ectosomes and exosomes with a size ranging from 0.1 to 1 μm, are key players in intercellular signaling. These EVs may carry specific proteins, mRNAs and microRNAs (miRNAs. Transfer of exosomes to neurons was shown to be mediated by oligodendrocytes, microglia and astrocytes that may either be supportive to neurons, or instead disseminate the disease. Interestingly, several recent reports have identified changes in miRNAs in depressed patients, which target not only crucial pathways associated with synaptic plasticity, learning and memory but also the production of neurotrophic factors and immune cell modulation. In this article, we discuss the role of neuroinflammation in the emergence of depression, namely dynamic alterations in the status of microglia response to stimulation, and how their activation phenotypes may have an etiological role in neurodegeneneration, in particular in depressive-like behavior. We will overview the involvement of miRNAs, exosomes, ectosomes and microglia in regulating

  18. Proteomic and activity profiles of ascorbate-glutathione cycle enzymes in germinating barley embryo

    DEFF Research Database (Denmark)

    Bønsager, Birgit Christine; Shahpiri, Azar; Finnie, Christine

    2010-01-01

    Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity...

  19. Placental iodothyronine deiodinase III and II ratios, mRNA expression compared to enzyme activity

    NARCIS (Netherlands)

    Stulp, M. R.; de Vijlder, J. J.; Ris-Stalpers, C.

    1998-01-01

    Iodothyronine deiodinases III and II (D3 and D2) specific enzyme activities in human placenta both decrease with gestational age. The relation of the enzyme activities with their respective mRNA expression was investigated by semi-quantitative RT-PCR on human placenta mRNA. To investigate if RT-PCR

  20. Effects of ABO/Rh blood groups, G-6-P-D enzyme activity and ...

    African Journals Online (AJOL)

    Effects of ABO/Rh blood groups, G-6-P-D enzyme activity and haemoglobin. Theresa K Nkuo-Akenji, Paul Wepngong, Jane-Frances Akoachere. Abstract. The main objective was to investigate the effects of ABO/Rh blood groups, haemoglobin genotype and G-6-P-D enzyme activity on malaria. The study was carried out in ...

  1. Phytobiotic Utilization as Feed Additive in Feed for Pancreatic Enzyme Activity of Broiler Chicken

    Directory of Open Access Journals (Sweden)

    Sri Purwanti

    2015-09-01

    Full Text Available This research was conducted to evaluate the effect of turmeric water extract, garlic and combination turmeric and garlic as a feed additive in the broiler diet on pancreatic enzyme activity of broiler chicken. Effectivity of treatments was assessed by addition of phytobiotic (control, 015% zinc bacitracin, 2.5% TE, 2.0% GE, 2.5% TGE which were arranged Completely Randomized Design with 4 replications. The variables measured were pancreatic enzyme activity(amylase enzyme activity, protease enzyme activity  and lipase enzyme activity.The results showed that enzyme protein activity content of 2.5% TE supplementation is also high at 82.02 U/ml, then supplemented 2.5% TGE, 2.0% GE, negative control and positive control respectively 75.98 ; 72.02; 68.74; and 66.57 U/ml. The lipase enzyme activity whereas the negative control and a positive control differ significantly higher (P<0.05 to treatment with the addition of 2.5% TE, 2.0% GE and 2.5% TGE phytobiotic. The research concluded that the incorporation of 2.5% TE, 2% GE and combined 2.5% TGE as feed additive enhanced pancreatic enzyme activity.

  2. Pectic enzymes

    NARCIS (Netherlands)

    Benen, J.A.E.; Voragen, A.G.J.; Visser, J.

    2003-01-01

    The pectic enzymes comprise a diverse group of enzymes. They consist of main-chain depolymerases and esterases active on methyl- and acetylesters of galacturonosyl uronic acid residues. The depolymerizing enzymes comprise hydrolases as wel as lyases

  3. Drugs elevating extracellular adenosine administered in vivo induce serum colony-stimulating activity and interleukin-6 in mice

    Czech Academy of Sciences Publication Activity Database

    Weiterová, Lenka; Hofer, Michal; Pospíšil, Milan; Znojil, V.; Štreitová, Denisa

    2007-01-01

    Roč. 56, č. 4 (2007), s. 463-473 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GP305/03/D050 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : extracellular adenosine * serum colony-stimulating activity * interleukin-6 Subject RIV: BO - Biophysics Impact factor: 1.505, year: 2007

  4. Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

    Directory of Open Access Journals (Sweden)

    Brian Roland eLarsen

    2016-04-01

    Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

  5. Assessment of some Hepatic Enzyme activities in adult rabbits ...

    African Journals Online (AJOL)

    Therapeutic potentials of Garcinia kola (G. kola) have been extensively documented and several researches have asserted its protective uniqueness against liver disorders/diseases. It is the aim of this study to assess the level of some enzyme involved in liver cellular integrity in rabbits chronically fed G. kola. To achieve this ...

  6. Chitinolytic enzymes from Clostridium aminovalericum: Activity screening and purification

    Czech Academy of Sciences Publication Activity Database

    Šimůnek, Jiří; Tishchenko, Galina; Rozhetsky, K.; Bartoňová, Hana; Kopečný, Jan; Hodrová, Blanka

    2004-01-01

    Roč. 49, č. 2 (2004), s. 194-198 ISSN 0015-5632 R&D Projects: GA ČR GA525/00/0984 Institutional research plan: CEZ:AV0Z5045916 Keywords : chitinolytic enzyme Subject RIV: EE - Microbiology, Virology Impact factor: 1.034, year: 2004

  7. Decrease in Activities of Selected Rat Liver Enzymes following ...

    African Journals Online (AJOL)

    The effects of the chemical effluent from Soap and Detergent Industry on some rat liver enzymes were investigated. Chemical analyses of both the effluent and tap water which served as the control were carried out before various concentrations of the effluent (5%v/v, 25%v/v, 50%v/v and 100%v/v) were made. The effluent ...

  8. HPRT Enzyme Activity of Blood Cells From Patients With Downs Syndrome

    International Nuclear Information System (INIS)

    Sbubber, E.K.; Abdul-Rahman, M.H.; Sultan, A.F.; Hamamy, H.A.

    1998-01-01

    Hypoxanthine phosphoribosyl transferase (HPRT) enzyme activity was determined in erythrocytes from 16 children (aged below one year to 11 year) with down s syndrome using 8-C 14 Hypoxanthine and radioeleelrophorsis techniques. Significant (P<0.01) reduction in HPRT enzyme activity was seen in D S children compared to that of 18 (age and sex matched) healthy children. Pure 21 - trisomic erythrocytes expressed lower enzyme activity than mosaic cell. Mothers of D S children showed significantly (P<0.01) lower enzyme activity than mothers of normal children . Reduced activity of HPRT enzyme was also observed in PHA-stimulated lymphocytes of DS children and their mothers. These results indicated that deficiency of HPRT in D S patients may contribute to the abnormal purine metabolism associated with the symptomatology of this syndrome

  9. Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis.

    Science.gov (United States)

    Hagopian, Kevork; Tomilov, Alexey A; Tomilova, Natalia; Kim, Kyoungmi; Taylor, Sandra L; Lam, Adam K; Cortopassi, Gino A; McDonald, Roger B; Ramsey, Jon J

    2012-12-01

    ShcKO mice have low body fat and resist weight gain on a high fat diet, indicating that Shc proteins may influence enzymes involved in β-oxidation. To investigate this idea, the activities of β-oxidation and ketone body metabolism enzymes were measured. The activities of β-oxidation enzymes (acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and ketoacyl-CoA thiolase) in liver and hindlimb skeletal muscle, ketolytic enzymes (acetoacetyl-CoA thiolase, β-hydroxybutyrate dehydrogenase and 3-oxoacid-CoA transferase) in skeletal muscle, and ketogenic enzymes (acetoacetyl-CoA thiolase and β-hydroxybutyrate dehydrogenase) in liver were measured from wild-type and ShcKO mice. The activities of β-oxidation enzymes were increased (P<.05) in the ShcKO compared to wild-type mice in the fasted but not the fed state. In contrast, no uniform increases in the ketolytic enzyme activities were observed between ShcKO and wild-type mice. In liver, the activities of ketogenic enzymes were increased (P<.05) in ShcKO compared to wild-type mice in both the fed and fasted states. Levels of phosphorylated hormone sensitive lipase from adipocytes were also increased (P<.05) in fasted ShcKO mice. These studies indicate that the low Shc levels in ShcKO mice result in increased liver and muscle β-oxidation enzyme activities in response to fasting and induce chronic increases in the activity of liver ketogenic enzymes. Decreases in the level of Shc proteins should be considered as possible contributors to the increase in activity of fatty acid oxidation enzymes in response to physiological conditions which increase reliance on fatty acids as a source of energy. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.

    Directory of Open Access Journals (Sweden)

    Ziru Niu

    Full Text Available Extracellular vesicles (EVs are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC, ultra-filtration (UF, polymer-based precipitation (PBP, and PBP with size-based purification (PBP+SP for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF. Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05. Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05. All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05. The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.

  11. Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

    Science.gov (United States)

    Arez, Bruno F; Alves, Luís; Paixão, Susana M

    2014-11-01

    The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

  12. Mini Review: Basic Physiology and Factors Influencing Exogenous Enzymes Activity in the Porcine Gastrointestinal Tract

    DEFF Research Database (Denmark)

    Strube, Mikael Lenz; Meyer, Anne S.; Boye, Mette

    2013-01-01

    activity during intestinal transit are few, it is known that the enzymes, being protein molecules, can be negatively affected by the gastrointestinal proteolytic enzymes and the low pH in the stomach ventricle. In this review, the pH-values, endogenous proteases and other factors native to the digestive......The addition of exogenous enzymes to pig feed is used to enhance general nutrient availability and thus increase daily weight gain per feed unit. The enzymes used are mainly beta-glucanase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) and phytase (EC 3.1.3.8). Although in vivo data assessing feed enzyme...... tract of the adult pig and the piglet are discussed in relation to the stability of exogenous feed enzymes. Development of more consistent assessment methods which acknowledge such factors is warranted both in vitro and in vivo for proper evaluation and prediction of the efficiency of exogenous enzymes...

  13. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora

    2012-01-01

    calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca(2+) pathway. The resulting increase in the intracellular calcium concentration......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  14. Effects of metal ions on stability and activity of hyperthermophilic pyrolysin and further stabilization of this enzyme by modification of a Ca2+-binding site.

    Science.gov (United States)

    Zeng, Jing; Gao, Xiaowei; Dai, Zheng; Tang, Bing; Tang, Xiao-Feng

    2014-05-01

    Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na(+), Ca(2+), or Mg(2+) salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca(2+)-binding sites (Ca1 and Ca2) revealed that the binding of Ca(2+) is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca(2+)-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin.

  15. Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca2+-Binding Site

    Science.gov (United States)

    Zeng, Jing; Gao, Xiaowei; Dai, Zheng; Tang, Bing

    2014-01-01

    Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na+, Ca2+, or Mg2+ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca2+-binding sites (Ca1 and Ca2) revealed that the binding of Ca2+ is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca2+-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin. PMID:24561589

  16. Diversity and Enzyme Activity of Ectomycorrhizal Fungal Communities Following Nitrogen Fertilization in an Urban-Adjacent Pine Plantation

    Directory of Open Access Journals (Sweden)

    Chen Ning

    2018-02-01

    Full Text Available Rapid economic development and accelerated urbanization in China has resulted in widespread atmospheric nitrogen (N deposition. One consequence of N deposition is the alteration of mycorrhizal symbioses that are critical for plant resource acquisition (nitrogen, N, phosphorus, P, water. In this study, we characterized the diversity, composition, and functioning of ectomycorrhizal (ECM fungal communities in an urban-adjacent Pinus elliottii plantation under ambient N deposition (~24 kg N ha−1 year−1, and following N fertilization (low N, 50 kg N ha−1 year−1; high N, 300 kg N ha−1 year−1. ECM functioning was expressed as the potential activities of extracellular enzymes required for organic N (protease, P (phosphomonoesterase, and recalcitrant polymers (phenol oxidase. Despite high ambient N deposition, ECM community composition shifted under experimental N fertilization, and those changes were linked to disparate levels of soil minerals (P, K and organic matter (but not N, a decline in acid phosphatase (AP, and an increase in phenol oxidase (PO potential activities. Based on enzyme stoichiometry, medium-smooth exploration type ECM species invested more in C acquisition (PO relative to P (AP following high N fertilization than other exploration types. ECM species with hydrophilic mantles also showed higher enzymatic PO:AP ratios than taxa with hydrophobic mantles. Our findings add to the accumulating evidence that shifts in ECM community composition and taxa specialized in organic C, N, and P degradation could modulate the soil nutrient cycling in forests exposed to chronic elevated N input.

  17. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise

    Directory of Open Access Journals (Sweden)

    Y Hu

    2015-09-01

    Full Text Available The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting exercise. Dopamine (DA and 5-hydroxytryptamine (5-HT levels were subsequently detected with high-performance liquid chromatography (HPLC. For immunohistochemistry study, the expression of DRD 2 and HT 2C receptors in the STN, before, immediately after and 90 min after exhaustion was detected through immunohistochemistry technique. Microdialysis study results showed that the extracellular DA and 5-HT neurotransmitters increased significantly throughout the procedure of exhausting exercise and the recovery period (P0.05. Our results suggest that the increased extracellular DA and 5-HT in the STN might be one important factor leading to the enhanced STN neural activity and the development of fatigue during exhausting exercise. This study may essentially offer useful evidence for better understanding of the mechanism of the central type of exercise-induced fatigue.

  18. Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

    Science.gov (United States)

    Nadar, Shamraja S; Rathod, Virendra K

    2017-08-22

    Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

  19. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    . The negative effect of BSA on enzyme was not observed before. The residual activity of horseradish peroxidase was also improved by the reconstituted skim milk: addition of reconstituted skim milk prior to heat treatment resulted in higher residual activity of HRP compared to no addition (58±3% and 30......±1%, respectively) The findings of this study show that β-casein can influence the response of food enzymes to heat treatment. β-Casein is not a universal chaperone and its effect on different targets needs to be evaluated on a case-by-case basis. This study also shows that proteins as e.g. BSA may affect......ABSTRACT Activity of endogenous enzymes may cause browning of fruits and vegetables. These enzymes can be inactivated, for example by heat treatment, but the response of enzymes to heat treatment depends on many factors. Foods are very complex systems and the stability of enzymes...

  20. Preparation and characterization of a novel extracellular polysaccharide with antioxidant activity, from the mangrove-associated fungus Fusarium oxysporum.

    Science.gov (United States)

    Chen, Yan-Li; Mao, Wen-Jun; Tao, Hong-Wen; Zhu, Wei-Ming; Yan, Meng-Xia; Liu, Xue; Guo, Tian-Tian; Guo, Tao

    2015-04-01

    Marine fungi are recognized as an abundant source of extracellular polysaccharides with novel structures. Mangrove fungi constitute the second largest ecological group of the marine fungi, and many of them are new or inadequately described species and may produce extracellular polysaccharides with novel functions and structures that could be explored as a source of useful polymers. The mangrove-associated fungus Fusarium oxysporum produces an extracellular polysaccharide, Fw-1, when grown in potato dextrose-agar medium. The homogeneous Fw-1 was isolated from the fermented broth by a combination of ethanol precipitation, ion-exchange, and gel filtration chromatography. Chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance spectroscopies showed that Fw-1 consisted of galactose, glucose, and mannose in a molar ratio of 1.33:1.33:1.00, and its molecular weight was about 61.2 kDa. The structure of Fw-1 contains a backbone of (1 → 6)-linked β-D-galactofuranose residues with multiple side chains. The branches consist of terminal α-D-glucopyranose residues, or short chains containing (1 → 2)-linked α-D-glucopyranose, (1 → 2)-linked β-D-mannopyranose, and terminal β-D-mannopyranose residues. The side chains are connected to C-2 of galactofuranose residues of backbone. The antioxidant activity of Fw-1 was evaluated with the scavenging abilities on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro, and the results indicated that Fw-1 possessed good antioxidant activity, especially the scavenging ability on hydroxyl radicals. The investigation demonstrated that Fw-1 is a novel galactofuranose-containing polysaccharide with different structural characteristics from extracellular polysaccharides from other marine microorganisms and could be a potential source of antioxidant.

  1. Monocyte activation drives preservation of membrane thiols by promoting release of oxidised membrane moieties via extracellular vesicles.

    Science.gov (United States)

    Szabó-Taylor, K É; Tóth, E Á; Balogh, A M; Sódar, B W; Kádár, L; Pálóczi, K; Fekete, N; Németh, A; Osteikoetxea, X; Vukman, K V; Holub, M; Pállinger, É; Nagy, Gy; Winyard, P G; Buzás, E I

    2017-07-01

    The redox state of cellular exofacial molecules is reflected by the amount of available thiols. Furthermore, surface thiols can be considered as indicators of immune cell activation. One group of thiol containing proteins, peroxiredoxins, in particular, have been associated with inflammation. In this study, we assessed surface thiols of the U937 and Thp1 monocyte cell lines and primary monocytes in vitro upon inflammatory stimulation by irreversibly labelling the cells with a fluorescent derivative of maleimide. We also investigated exofacial thiols on circulating blood mononuclear cells in patients with rheumatoid arthritis and healthy controls. When analysing extracellular vesicles, we combined thiol labelling with the use of antibodies to specific CD markers to exclude extracellular vesicle mimicking signals from thiol containing protein aggregates. Furthermore, differential detergent lysis was applied to confirm the vesicular nature of the detected extracellular events in blood plasma. We found an increase in exofacial thiols on monocytes upon in vitro stimulation by LPS or TNF, both in primary monocytes and monocytic cell lines (pextracellular vesicles showed a decrease in their exofacial thiols compared with those from unstimulated cells (pextracellular vesicles of isolated CD14 + cells from rheumatoid arthritis patients had decreased thiol levels compared with healthy subjects (pextracellular vesicles was increased in rheumatoid arthritis blood plasma (pextracellular vesicle-enriched preparations from blood plasma. Our data show that cell surface thiols play a protective role and reflect oxidative stress resistance state in activated immune cells. Furthermore, they support a role of extracellular vesicles in the redox regulation of human monocytes, possibly representing an antioxidant mechanism. Copyright © 2017. Published by Elsevier Inc.

  2. Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates.

    Science.gov (United States)

    Peterson, R A; Bradner, J R; Roberts, T H; Nevalainen, K M H

    2009-02-01

    Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest. Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25 degrees C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15 degrees C and some at 39 degrees C. Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates. To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.

  3. Glutathione peroxidase 3 localizes to the epithelial lining fluid and the extracellular matrix in interstitial lung disease

    OpenAIRE

    Schamberger, A.; Schiller, H.; Fernandez, I.; Sterclova, M.; Heinzelmann, K.; Hennen, E.; Hatz, R.; Behr, J.; Vasakova, M.; Mann, M.; Eickelberg, O.; Staab-Weijnitz, C.

    2016-01-01

    Aberrant antioxidant activity and excessive deposition of extracellular matrix (ECM) are hallmarks of interstitial lung diseases (ILD). It is known that oxidative stress alters the ECM, but extracellular antioxidant defence mechanisms in ILD are incompletely understood. Here, we extracted abundance and detergent solubility of extracellular antioxidant enzymes from a proteomic dataset of bleomycin-induced lung fibrosis in mice and assessed regulation and distribution of glutathione peroxidase ...

  4. Enzyme Stability and Activity in Non-Aqueous Reaction Systems: A Mini Review

    Directory of Open Access Journals (Sweden)

    Shihui Wang

    2016-02-01

    Full Text Available Enormous interest in biocatalysis in non-aqueous phase has recently been triggered due to the merits of good enantioselectivity, reverse thermodynamic equilibrium, and no water-dependent side reactions. It has been demonstrated that enzyme has high activity and stability in non-aqueous media, and the variation of enzyme activity is attributed to its conformational modifications. This review comprehensively addresses the stability and activity of the intact enzymes in various non-aqueous systems, such as organic solvents, ionic liquids, sub-/super-critical fluids and their combined mixtures. It has been revealed that critical factors such as Log P, functional groups and the molecular structures of the solvents define the microenvironment surrounding the enzyme molecule and affect enzyme tertiary and secondary structure, influencing enzyme catalytic properties. Therefore, it is of high importance for biocatalysis in non-aqueous media to elucidate the links between the microenvironment surrounding enzyme surface and its stability and activity. In fact, a better understanding of the correlation between different non-aqueous environments and enzyme structure, stability and activity can contribute to identifying the most suitable reaction medium for a given biotransformation.

  5. Cell-specific extracellular phosphatase activity of dinoflagellate populations in acidified mountain lakes

    Czech Academy of Sciences Publication Activity Database

    Novotná, J.; Nedbalová, Linda; Kopáček, Jiří; Vrba, Jaroslav

    2010-01-01

    Roč. 46, č. 4 (2010), s. 635-644 ISSN 0022-3646 R&D Projects: GA AV ČR IAA600170602; GA ČR GA206/07/1200 Institutional research plan: CEZ:AV0Z60050516; CEZ:AV0Z60170517 Keywords : extracellular phosphatases * acidified lakes * dinoflagellates Subject RIV: EF - Botanics Impact factor: 2.239, year: 2010

  6. Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

    DEFF Research Database (Denmark)

    Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern

    2012-01-01

    Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional....... Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...

  7. Antifungal activity and expression patterns of extracellular chitinase and β-1,3-glucanase in Wickerhamomyces anomalus EG2 treated with chitin and glucan.

    Science.gov (United States)

    Hong, Sin-Hyoung; Song, Yong-Su; Seo, Dong-Jun; Kim, Kil-Yong; Jung, Woo-Jin

    2017-09-01

    In this study, the expression patterns of extracellular chitinase and β-1,3-glucanase from cultured Wickerhamomyces anomalus EG2 treated with chitin, glucan, and chemical chitinase inhibitors (kinetin, caffeine, and acetazolamide) were investigated using SDS-PAGE. Relationship between enzyme expression and antifungal activity from yeast plays a very important role for biocontrol of phytopathoges. To determine antifungal activity against phytopathogens, W. anomalus EG2 was shown to strongly inhibit hyphal growth of Fusarium oxysporum KACC 40032 and Rhizoctonia solani KACC 40111. Slight chitinase activity was observed 12 h after incubation in both PDB and YPD medium without colloidal chitin. The molecular weight of chitinase was approximately 124 kDa β-1,3-Glucanase isoenzyme (GN1 and GN2) was observed distinctly on SDS-PAGE gels when laminarin was used as a substrate. β-1,3-Glucanase isoenzyme was not observed when using glucan-containing high polymer complex (GHPC) as a substrate. Production of chitinase from W. anomalus EG2 was inhibited slightly by acetazolamide. Abnormal and cluster-shaped cells of W. anomalus EG2 were observed in both PDB and YPD medium treated with colloidal chitin. These results indicated that W. anomalus EG2 could be applied commercially as a biological control agent of phytopathogens and as a bioinhibitor of yeast cell growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    Science.gov (United States)

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Inhibitory activity of Plantago major L. on angiotensin I-converting enzyme.

    Science.gov (United States)

    Nhiem, Nguyen Xuan; Tai, Bui Huu; Van Kiem, Phan; Van Minh, Chau; Cuong, Nguyen Xuan; Tung, Nguyen Huu; Thu, Vu Kim; Trung, Trinh Nam; Anh, Hoang Le Tuan; Jo, Sung-Hoon; Jang, Hae-Dong; Kwon, Young-In; Kim, Young Ho

    2011-03-01

    Eight compounds were isolated from methanol extract of Plantago major L. leaves and investigated for their ability to inhibit angiotensin I-converting enzyme activity. Among them, compound 1 showed the most potent inhibition with rate of 28.06 ± 0.21% at a concentration of 100 μM. Compounds 2 and 8 exhibited weak activities. These results suggest that compound 1 might contribute to the ability of P. major to inhibit the activity of angiotensin I- converting enzyme.

  10. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)

    ROZEBOOM, HJ; BUDIANI, A; BEINTEMA, JJ

    1990-01-01

    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To

  11. Increased activities of mitochondrial enzymes in white adipose tissue in trained rats

    DEFF Research Database (Denmark)

    Stallknecht, B; Vinten, J; Ploug, T

    1991-01-01

    of 8-12 rats were swim trained for 10 wk or served as either sedentary, sham swim-trained, or cold-stressed controls. White adipose tissue was removed, and the activities of the respiratory chain enzyme cytochrome-c oxidase (CCO) and of the enzyme malate dehydrogenase (MDH), which participates...

  12. Exo-inulinase of Aspergillus niger N402: A hydrolytic enzyme with significant transfructosylating activity

    NARCIS (Netherlands)

    Goosen, C.; Maarel, M.J. E.C. van der; Dijkhuizen, L.

    2008-01-01

    The purified exo-inulinase enzyme of Aspergillus niger N402 (AngInuE; heterologously expressed in Escherichia coli) displayed a sucrose:inulin (S/I) hydrolysis ratio of 2.3, characteristic for a typical exo-inulinase. The enzyme also had significant transfructosylating activity with increasing

  13. Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

    Science.gov (United States)

    The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

  14. Effect of citric acid and microbial phytase on serum enzyme activities ...

    African Journals Online (AJOL)

    Effect of citric acid and microbial phytase on serum enzyme activities and plasma minerals retention in broiler chicks. ... African Journal of Biotechnology ... An experiment was conducted to study the effect of microbial phytase supplementation and citric acid in broiler chicks fed corn-soybean meal base diets on enzyme ...

  15. Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

    Science.gov (United States)

    Kondo, Ayano; Yamamoto, Shogo; Nakaki, Ryo; Shimamura, Teppei; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Yoshida, Tetsuo; Aburatani, Hiroyuki; Osawa, Tsuyoshi

    2017-02-28

    Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme...... specificity and acting as an allosteric site. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. Despite a relatively long history, it is only in recent years that SBSs have been studied in great...

  17. Pyrrolizidine alkaloids--genotoxicity, metabolism enzymes, metabolic activation, and mechanisms.

    Science.gov (United States)

    Fu, Peter P; Xia, Qingsu; Lin, Ge; Chou, Ming W

    2004-02-01

    Pyrrolizidine alkaloid-containing plants are widely distributed in the world and are probably the most common poisonous plants affecting livestock, wildlife, and humans. Because of their abundance and potent toxicities, the mechanisms by which pyrrolizidine alkaloids induce genotoxicities, particularly carcinogenicity, were extensively studied for several decades but not exclusively elucidated until recently. To date, the pyrrolizidine alkaloid-induced genotoxicities were revealed to be elicited by the hepatic metabolism of these naturally occurring toxins. In this review, we present updated information on the metabolism, metabolizing enzymes, and the mechanisms by which pyrrolizidine alkaloids exert genotoxicity and tumorigenicity.

  18. Effect of different growth parameters on chitinase enzyme activity ...

    African Journals Online (AJOL)

    Optimization of culture conditions revealed that the enzyme production was maximum in pH 7.5 (107.4 ± 0.50 U/ml), temperature 35°C (103.15 ± 1.74 U/ml) when the carbon and the nitrogen sources used were CMC (106.0 ± 1.89 U/ml) and KNO3 (91.2 ± 1.51 U/ml), respectively. The total chitinase production for all optimum ...

  19. An Experimental Insight into Extracellular Phosphatases – Differential Induction of Cell-Specific Activity in Green Algae Cultured under Various Phosphorus Conditions

    Directory of Open Access Journals (Sweden)

    Jaroslav Vrba

    2018-02-01

    Full Text Available Extracellular phosphatase activity (PA has been used as an overall indicator of P depletion in lake phytoplankton. However, detailed insights into the mechanisms of PA regulation are still limited, especially in the case of acid phosphatases. The novel substrate ELF97 phosphate allows for tagging PA on single cells in an epifluorescence microscope. This fluorescence-labeled enzyme activity (FLEA assay enables for autecological studies in natural phytoplankton and algal cultures. We combined the FLEA assay with image analysis to measure cell-specific acid PA in two closely related species of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta isolated from two acidic lakes with distinct P availability. The strains were cultured in a mineral medium supplied with organic (beta-glycerol phosphate or inorganic (orthophosphate P at three concentrations. Both strains responded to experimental conditions in a similar way, suggesting that acid extracellular phosphatases were regulated irrespectively of the origin and history of the strains. We found an increase in cell-specific PA at low P concentration and the cultures grown with organic P produced significantly higher (ca. 10-fold PA than those cultured with the same concentrations of inorganic P. The cell-specific PA measured in the cultures grown with the lowest organic P concentration roughly corresponded to those of the original Coccomyxa population from an acidic lake with impaired P availability. The ability of Coccomyxa strains to produce extracellular phosphatases, together with tolerance for both low pH and metals can be one of the factors enabling the dominance of the genus in extreme conditions of acidic lakes. The analysis of frequency distribution of the single-cell PA documented that simple visual counting of ‘active’ (labeled and ‘non-active’ (non-labeled cells can lead to biased conclusions regarding algal P status because the actual PA of the ‘active’ cells can vary from

  20. Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Fukushima, Tadamasa [Shimadzu GLC Ltd., Phenomenex Support Centre, Tokyo 110-0016 (Japan); Morimoto, Hisao; Usami, Ron [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Maekawa, Toru, E-mail: maekawa@toyonet.toyo.ac.jp [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan)

    2010-03-19

    We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

  1. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    Science.gov (United States)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  2. An appraisal of the enzyme stability-activity trade-off.

    Science.gov (United States)

    Miller, Scott R

    2017-07-01

    A longstanding idea in evolutionary physiology is that an enzyme cannot jointly optimize performance at both high and low temperatures due to a trade-off between stability and activity. Although a stability-activity trade-off has been observed for well-characterized examples, such a trade-off is not imposed by any physical chemical constraint. To better understand the pervasiveness of this trade-off, I investigated the stability-activity relationship for comparative biochemical studies of purified orthologous enzymes identified by a literature search. The nature of this relationship varied greatly among studies. Notably, studies of enzymes with low mean synonymous nucleotide sequence divergence were less likely to exhibit the predicted negative correlation between stability and activity. Similarly, a survey of directed evolution investigations of the stability-activity relationship indicated that these traits are often uncoupled among nearly identical yet phenotypically divergent enzymes. This suggests that the presumptive trade-off often reported for investigations of enzymes with high mean sequence divergence may in some cases instead be a consequence of the degeneration over time of enzyme function in unselected environments, rather than a direct effect of thermal adaptation. The results caution against the general assertion of a stability-activity trade-off during enzyme adaptation. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

  3. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation

    Science.gov (United States)

    Styrczewska, Monika; Kostyn, Anna; Kulma, Anna; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Prescha, Anna; Czuj, Tadeusz; Szopa, Jan

    2015-01-01

    Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification. PMID:26347154

  4. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation

    Directory of Open Access Journals (Sweden)

    Monika Styrczewska

    2015-01-01

    Full Text Available Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD, phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.

  5. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation.

    Science.gov (United States)

    Styrczewska, Monika; Kostyn, Anna; Kulma, Anna; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Prescha, Anna; Czuj, Tadeusz; Szopa, Jan

    2015-01-01

    Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.

  6. Comparative evaluation of enzyme activities and phenol content of ...

    African Journals Online (AJOL)

    Interestingly, biochemical analyses showed the highest phenolic content and PME activity in plants treated with EM Bokashi. All treatments significantly increased POX activity while they decreased PPO activity. In addition, significant and positive correlations (P < 0.01) were observed between stem length and PME activity ...

  7. Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

    Science.gov (United States)

    Martín-Estebané, María; Navascués, Julio; Sierra-Martín, Ana; Martín-Guerrero, Sandra M; Cuadros, Miguel A; Carrasco, María-Carmen; Marín-Teva, José L

    2017-01-01

    Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the

  8. Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences.

    Science.gov (United States)

    Schwarzenberger, Anke; Fink, Patrick

    2018-04-01

    Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and β-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wosten-van Asperen, Roelie M.; Bos, Albert; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, Rene

    2013-01-01

    Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts

  10. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wösten-van Asperen, Roelie M.; Bos, Albert P.; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, René

    2013-01-01

    Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin

  11. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

    International Nuclear Information System (INIS)

    Beckman, J.S.; Minor, R.L. Jr.; White, C.W.; Repine, J.E.; Rosen, G.M.; Freeman, B.A.

    1988-01-01

    Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from 125 I-PEG-catalase or 125 I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species

  12. Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

    Science.gov (United States)

    Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

    2013-04-01

    A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

    Directory of Open Access Journals (Sweden)

    Hyong Woo Choi

    2016-03-01

    Full Text Available Damage-associated molecular pattern molecules (DAMPs signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3 is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i MAPK activation, ii defense-related gene expression, iii callose deposition, and iv enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast. Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA, which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

  14. Mining anaerobic digester consortia metagenomes for secreted carbohydrate active enzymes

    DEFF Research Database (Denmark)

    Wilkens, Casper; Busk, Peter Kamp; Pilgaard, Bo

    , and food wastes (Alvarado et al., 2014). The processes and the roles of the microorganisms that are involved in biomass conversion and methane production in ADs are still not fully understood. We are investigating thermophilic and mesophilic ADs that use wastewater surplus sludge for methane production......Anaerobic digesters (ADs) are one of several ways to produce renewable energy, which in the case of ADs is in the form of methane. Several microbial groups are involved in anaerobic degradation of organic wastes such as animal manures and wastewater, and solid organic wastes such as sludge, crop...... was done with the Peptide Pattern Recognition (PPR) program (Busk and Lange, 2013), which is a novel non-alignment based approach that can predict function of e.g. CAZymes. PPR identifies a set of short conserved sequences, which can be used as a finger print when mining genomes for novel enzymes. In both...

  15. ENZYME ACTIVITIES OF PADDY SOILS AND RELATIONSHIPS WITH THE SOIL PROPERTIES

    Directory of Open Access Journals (Sweden)

    Rıdvan KIZILKAYA

    1998-03-01

    Full Text Available This study was carried out to determine the effect of soil properties on enzyme activities of paddy soils, the sample of which were taken from Üçpınar, Harız, Doğancı, Kaygusuz, Emenli, Sarıköy and Gelemenağarı villages where rice cultivation is an intensive agricultural system. In this study, soil properties having effects on urease, phosphatase, ß-glucosidase and catalase enzyme activities were setforth. Urease enzyme activities of soil samples varied from 24.12 to 39.03 mg N 100 g dry soil -1 . Significant correlations were determined between urease enzyme activities and organic matter (r = 0.89**, extractable Mn (r = 0.74**, exchangable K (r = 0.73** and total P content of soil (r = 0.81*. Acid phosphatase enzyme activity varied between 3.00-17.44 mg phenol 100 g dry soil -1 , alkaline phosphatase enzyme activity between 12.00-25.53 mg phenol 100 g dry soil-1 . Exchangable Mg (r = 0.71* and extractable Cu (r = 0.74* were found to have positive effect on acid phosphatase enzyme activity and pH (r = 0.73*, exchangable Ca (r = 0.74*, exchangable Mg (r = 0.71*, exchangable total basic cations (r = 0.79* and extractable Cu (r = 0.70* had positive effects on alkaline phosphatase enzyme activity, whereas total P (r = - 0.84** affected the activity negatively. ß-glucosidase enzyme activity was measured to vary between 1.12-3.64 mg salingen 100 g dry soil -1 . It was also observed that extractable Zn content of soil samples (r = - 0.97** had negative effect on ß-glucosidase activity, wheras total exchangable acidic cations (r = 0.70* affected the activity positively. Catalase enzyme activities of soils changed between 5.25 - 9.00 mg O2 5 g dry soil -1 . Significant correlations were found between catalase activities and fraction of soils and extractable Fe content. Positive correlations, however, were determined between catalase activities and clay fraction (r = 0.82* and salt content (r = 0.83** of samples.

  16. Recovery of active pathogenesis-related enzymes from the apoplast ...

    African Journals Online (AJOL)

    Overall protease activity intensity was higher in the symplast. Maximum symplast contamination of the apoplast was 2% as estimated by glucose 6-phosphate dehydrogenase activity, a biochemical marker for symplast. Accumulation of pathogenesis-related enzymatic activities in the apoplast of M. acuminata leaf tissue was ...

  17. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Energy Technology Data Exchange (ETDEWEB)

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  18. Definition of regional dependence of activity antioxidative enzymes means of the dispersive analysis

    Directory of Open Access Journals (Sweden)

    Anatoly T. Bykov

    2011-05-01

    Full Text Available In article application of the dispersive analysis for an estimation of dependence of activity antioxidative enzymes from region of constant residing, age, sex and the disease diagnosis is considered.

  19. Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

    Science.gov (United States)

    Gür, Sinem Diken; İdil, Neslihan; Aksöz, Nilüfer

    2018-02-01

    In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl 2 concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

  20. Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

    OpenAIRE

    Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

    2005-01-01

    In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocon...

  1. Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

    Science.gov (United States)

    Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

    2005-01-01

    In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol. PMID:16151170

  2. Climate effect on soil enzyme activities and dissolved organic carbon in mountain calcareous soils: a soil-transplant experiment

    Science.gov (United States)

    Puissant, Jérémy; Cécillon, Lauric; Mills, Robert T. E.; Gavazov, Konstantin; Robroek, Bjorn J. M.; Spiegelberger, Thomas; Buttler, Alexandre; Brun, Jean-Jacques

    2013-04-01

    Mountain soils store huge amounts of carbon as soil organic matter (SOM) which may be highly vulnerable to the strong climate changes that mountain areas currently experience worldwide. Climate modifications are expected to impact microbial activity which could change the rate of SOM decomposition/accumulation, thereby questioning the net C source/sink character of mountain soils. To simulate future climate change expected in the 21st century in the calcareous pre-Alps, 15 blocks (30 cm deep) of undisturbed soil were taken from a mountain pasture located at 1400 m a.s.l. (Marchairuz, Jura, Switzerland) and transplanted into lysimeters at the same site (control) and at two other sites located at 1000 m a.s.l. and 600 m a.s.l. (5 replicates per site). This transplantation experiment which started in 2009 simulates a climate warming with a temperature increase of 4° C and a decreased humidity of 40 % at the lowest site. In this study, we used soil extracellular enzyme activities (EEA) as functional indicators of SOM decomposition to evaluate the effect of climate change on microbial activity and SOM dynamics along the seasons. Dissolved organic carbon (DOC) was also measured to quantify the assimilable carbon for microorganism. In autumn 2012, a first sampling step out of four (winter, spring and summer 2013) has been realized. We extracted 15 cm deep soil cores from each transplant (x15) and measured (i) DOC and (ii) the activities of nine different enzymes. Enzymes were chosen to represent the degradation of the most common classes of biogeochemical compounds in SOM. β-glucosidase, β-D-cellubiosidase, β-Xylosidase, N-acetyl-β-glucosaminidase, leucine aminopeptidase, lipase, phenoloxidase respectively represented the degradation of sugar, cellulose, hemicellulose, chitin, protein, lipid and lignin. Moreover, the fluorescein diacetate (FDA) hydrolysis was used to provide an estimate of global microbial activity and phosphatase was used to estimate phosphorus

  3. REDUCED DNASE ENZYME ACTIVITY IN RESPONSE TO HIGH POST-INJURY MITOCHONDRIAL DNA CONCENTRATION PROVIDES A THERAPEUTIC TARGET FOR SIRS.

    Science.gov (United States)

    McIlroy, Daniel J; Minahan, Kyra; Keely, Simon; Lott, Natalie; Hansbro, Philip; Smith, Doug W; Balogh, Zsolt J

    2018-03-22

    Cell free mitochondrial DNA (mtDNA) is pro-inflammatory and has been detected in high concentrations in trauma patients' plasma. Deoxyribonuclease (DNase) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNase after major trauma is unknown. We hypothesized that DNase activity would be elevated after injury and trauma surgery, and would be associated with high concentrations of extracellular DNA. 2-year prospective study was performed on 103 consecutive trauma patients (Male 81%, Age: 38 IQR 30-59 years; ISS: 18 IQR 12-26) who underwent standardised major orthopaedic trauma surgical interventions. Blood was collected at 5 peri-operative time points (pre-op, post-op, 7hrs, 24hrs and 3 days post-operatively). Healthy control subjects (n=20) were also sampled. Cell free mtDNA and nuclear DNA(nDNA) was measured using quantitative polymerase chain reaction. DNase was also assayed in the same plasma samples. Increased levels of mtDNA (from Pre-op 163±86ng/ml to 3 days 282±201ng/ml pSIRS (p=0.026) but not MOF. The significant perioperative elevation in plasma free MtDNA concentration is associated with the development of SIRS. The fact that increased cell free DNA concentrations present with significantly lower than healthy control DNase activity suggests a potential therapeutic opportunity with DNase administration to modulate post-injury severe SIRS LEVEL OF EVIDENCE: Level II: Prospective comparative study.

  4. Modest induction of phase 2 enzyme activity in the F-344 rat prostate

    Directory of Open Access Journals (Sweden)

    Brooks James D

    2006-03-01

    Full Text Available Abstract Background Prostate cancer is the most commonly diagnosed malignancy in men and is thought to arise as a result of endogenous oxidative stress in the face of compromised carcinogen defenses. We tested whether carcinogen defense (phase 2 enzymes could be induced in the prostate tissues of rats after oral feeding of candidate phase 2 enzyme inducing compounds. Methods Male F344 rats were gavage fed sulforaphane, β-naphthoflavone, curcumin, dimethyl fumarate or vehicle control over five days, and on the sixth day, prostate, liver, kidney and bladder tissues were harvested. Cytosolic enzyme activities of nicotinamide quinone oxidoreductase (NQO1, total glutathione transferase (using DCNB and mu-class glutathione transferase (using CDNB were determined in the treated and control animals and compared. Results In prostatic tissues, sulforaphane produced modest but significant increases in the enzymatic activities of NQO1, total GST and GST-mu compared to control animals. β-naphthoflavone significantly increased NQO1 and GST-mu activities and curcumin increased total GST and GST-mu enzymatic activities. Dimethyl fumarate did not significantly increase prostatic phase 2 enzyme activity. Compared to control animals, sulforaphane also significantly induced NQO1 or total GST enzyme activity in the liver, kidney and, most significantly, in the bladder tissues. All compounds were well tolerated over the course of the gavage feedings. Conclusion Orally administered compounds will induce modestly phase 2 enzyme activity in the prostate although the significance of this degree of induction is unknown. The 4 different compounds also altered phase 2 enzyme activity to different degrees in different tissue types. Orally administered sulforaphane potently induces phase 2 enzymes in bladder tissues and should be investigated as a bladder cancer preventive agent.

  5. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

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    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  6. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study.

  7. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    Science.gov (United States)

    Semsang, Nuananong; Yu, LiangDeng

    2013-07-01

    Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29-60 keV and ion fluences of 1 × 1016 ions cm-2. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  8. Tree species composition influences enzyme activities and microbial biomass in the rhizosphere: a rhizobox approach.

    Science.gov (United States)

    Fang, Shengzuo; Liu, Dong; Tian, Ye; Deng, Shiping; Shang, Xulan

    2013-01-01

    Monoculture causes nutrient losses and leads to declines in soil fertility and biomass production over successive cultivation. The rhizosphere, a zone of usually high microbial activities and clearly distinct from bulk soil, is defined as the volume of soil around living roots and influenced by root activities. Here we investigated enzyme activities and microbial biomass in the rhizosphere under different tree compositions. Six treatments with poplar, willow, and alder mono- or mixed seedlings were grown in rhizoboxes. Enzyme activities associated with nitrogen cycling and microbial biomass were measured in all rhizosphere and bulk soils. Both enzyme activities and microbial biomass in the rhizosphere differed significantly tree compositions. Microbial biomass contents were more sensitive to the changes of the rhizosphere environment than enzyme activities. Tree species coexistence did not consistently increase tested enzyme activities and microbial biomass, but varied depending on the complementarities of species traits. In general, impacts of tree species and coexistence were more pronounced on microbial composition than total biomass, evidenced by differences in microbial biomass C/N ratios stratified across the rhizosphere soils. Compared to poplar clone monoculture, other tree species addition obviously increased rhizosphere urease activity, but greatly reduced rhizosphere L-asparaginase activity. Poplar growth was enhanced only when coexisted with alder. Our results suggested that a highly productive or keystone plant species in a community had greater influence over soil functions than the contribution of diversity.

  9. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xia Luo

    2015-01-01

    Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

  10. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

    Science.gov (United States)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  11. Scavenging activity, anti-inflammatory and diabetes related enzyme ...

    African Journals Online (AJOL)

    The total phenolic content measured by Folin-ciocalteu method. The raw leaf extracts of the selected varieties were found to contain a high content of total phenolic content (342.45 mg GAE/ gDW for GE) and therefore exhibited a higher antioxidant activity and inhibitory effect of radicals scavenging activity against DPPH and ...

  12. Changes in activities of enzymes of glutamate metabolism in rat ...

    African Journals Online (AJOL)

    ... acute treatment GAD activity was significantiy inhibited in ail the brain regions except in cerebral cortex where the inhibition was non-significant However, under sub-acute treatment GAD activity showed an elevation in cerebral cortex. cerebellum and striatum, whiie showing a decrease in hippocampus and ponsmedulla.

  13. Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

    Science.gov (United States)

    Desguin, Benoît; Goffin, Philippe; Viaene, Eric; Kleerebezem, Michiel; Martin-Diaconescu, Vlad; Maroney, Michael J; Declercq, Jean-Paul; Soumillion, Patrice; Hols, Pascal

    2014-01-01

    Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes. PMID:24710389

  14. Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

    Science.gov (United States)

    Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

    2014-12-14

    Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

  15. Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji'an, China.

    Science.gov (United States)

    Yin, Juxin; Zhang, Daihui; Zhuang, Jianjian; Huang, Yi; Mu, Ying; Lv, Shaowu

    2017-12-11

    Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.

  16. Nerve agent hydrolysis activity designed into a human drug metabolism enzyme.

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    Andrew C Hemmert

    2011-03-01

    Full Text Available Organophosphorus (OP nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.

  17. Milk enzyme activities and subclinical mastitis among women in Guinea-Bissau

    DEFF Research Database (Denmark)

    Rasmussen, Lill Brith Wium; Hartvig, Ditte Luise; Kæstel, Pernille

    2008-01-01

    Background: Subclinical mastititis (SCM) is a condition with raised milk concentration of sodium and milk immune factors. The milk enzymes N-acetyl-β-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), acid phosphatase (AcP), and alkaline phosphatase (AP) have attracted attention in dairy...... research as indicators of SCM, udder health, and milk quality. Study Design: To investigate if milk enzyme activities and the inflammatory interleukin 8 (IL-8) level are increased in women with SCM, we measured sodium, potassium, NAGase, LDH, AcP, AP, and IL-8 in breastmilk samples collected at 2 months...... in univariate linear regression (p enzymes and IL-8). Conclusions: A positive association between the Na/K ratio and the breastmilk enzymes NAGase, LDH, AcP, and AP was found. Breastmilk enzymes have not previously been investigated in relation to SCM in women, and further...

  18. Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

    NARCIS (Netherlands)

    Dunning, Sandra; Rehman, Atta Ur; Tiebosch, Marjolein H.; Hannivoort, Rebekka A.; Haijer, Floris W.; Woudenberg, Jannes; van den Heuvel, Fiona A. J.; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han

    2013-01-01

    Background: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated

  19. Mitochondrial respiratory and antioxidative enzyme activities in turkey meat.

    Science.gov (United States)

    Janisch, S; Wicke, M; Krischek, C

    2012-02-01

    Meat quality and (anti)oxidative metabolism of m. pectoralis superficialis (MPS), m. gastrocnemius (MG) and m. iliotibilialis lateralis (MIL) from turkey toms were analysed. After slaughter, pH of MPS and MG decreased and electrical conductivity of the MPS increased. The MG had generally higher pH values. The meat lightness (L) and redness (a) increased in MG and MPS after slaughter. The MPS always had higher L and lower a values. Mitochondrial respiratory activities (MRA) were higher in the MIL than the MPS. The activities of superoxide dismutase (SOD) and glutathione peroxidase, analysed in the MPS, increased and the glutathione reductase activity decreased after slaughter. Meat samples with lower pH24 h p.m. had higher drip loss and L values. The MRA were tendentially lower and the SOD activities higher in these samples. These results indicate a relation between the meat quality, the antioxidative metabolism and mitochondrial respiration.

  20. DNA topoisomerase II enzyme activity appears in mouse sperm ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Sperm suspensions of 4 male mice (A, B, C and D), having an initial motility grade of 3.5 were used to examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was 75%, male B was 80%, male C was 70% and male D was 60%. Top 2 activity was.

  1. DNA topoisomerase II enzyme activity appears in mouse sperm ...

    African Journals Online (AJOL)

    Sperm suspensions of 4 male mice (A, B, C and D), having an initial motility grade of 3.5 were used to examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was 75%, male B was 80%, male C was 70% and male D was 60%. Top 2 activity was examined by ...

  2. Stabilization of enzymes by multipoint covalent immobilization on supports activated with glyoxyl groups.

    Science.gov (United States)

    López-Gallego, Fernando; Fernandez-Lorente, Gloria; Rocha-Martin, Javier; Bolivar, Juan M; Mateo, Cesar; Guisan, Jose M

    2013-01-01

    Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization has to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of Lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

  3. Enzymes extracted from apple peels have activity in reducing higher alcohols in Chinese liquors.

    Science.gov (United States)

    Han, Qi'an; Shi, Junling; Zhu, Jing; Lv, Hongliang; Du, Shuangkui

    2014-10-01

    As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols.

  4. Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.

    Science.gov (United States)

    Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N

    2016-12-01

    Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.

  5. Novel TPP-riboswitch activators bypass metabolic enzyme dependency.

    Science.gov (United States)

    Lünse, Christina E; Scott, Fraser J; Suckling, Colin J; Mayer, Günter

    2014-01-01

    Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  6. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa.

    Directory of Open Access Journals (Sweden)

    Shoubing Wang

    Full Text Available Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA, and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05 after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01 from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01. The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice.

  7. Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

    Science.gov (United States)

    Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, José D.; Vargas, Aníbal A.; Verdugo, Christian A.; Jara, Oscar; Martínez, Agustín D.; Sáez, Juan C.

    2014-01-01

    Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects. PMID:25237294

  8. Gaseous environment of plants and activity of enzymes of carbohydrate catabolism

    International Nuclear Information System (INIS)

    Ivanov, B.F.; Zemlyanukhin, A.A.; Igamberdiev, A.U.; Salam, A.M.M.

    1989-01-01

    The authors investigated the action of hypoxia and high CO 2 concentration in the atmosphere on activity of phosphofructokinase, aldolase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, and isocitrate lyase in pea seedlings (Pisum sativum L.), corn scutella (Zea mays L.), and hemp cotyledons (Cannabis sativa L.). The first 4-12h of hypoxia witnessed suppression of enzymes of the initial stages of glycolysis (glucose-6-phosphate isomerase, phosphofructokinase)and activation of enzymes of its final stages (alcohol dehydrogenase and lactate dehydrogenase) and enzymes linking glycolysis and the pentose phosphate pathway (aldolase and glucose-6-phosphate dehydrogenase). An excess of CO 2 in the environment accelerated and amplified this effect. At the end of a 24-h period of anaerobic incubation, deviations of enzyme activity from the control were leveled in both gaseous environments. An exception was observed in the case of phosphofructokinase, whose activity increased markedly at this time in plants exposed to CO 2 . Changes in activity of the enzymes were coupled with changes in their kinetic parameters (apparent K m and V max values). The activity of isocitrate lyase was suppressed in both variants of hypoxic gaseous environments, a finding that does not agree with the hypothesis as to participation of the glyoxylate cycle in the metabolic response of plants to oxygen stress. Thus, temporary inhibition of the system of glycolysis and activation of the pentose phosphate pathway constituted the initial response of the plants to O 2 stress, and CO 2 intensified this metabolic response

  9. Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

    Directory of Open Access Journals (Sweden)

    Azevedo R.B.

    2001-01-01

    Full Text Available Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GSH-Px were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%. Removal of the gonads in both males and females (comparison between castrated groups increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48% CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

  10. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  11. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens.

    Directory of Open Access Journals (Sweden)

    Johanna Rytioja

    Full Text Available White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.

  12. Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography.

    Science.gov (United States)

    Hughes, Alex J; Herr, Amy E

    2010-05-01

    We describe a sensitive zymography technique that utilizes an automated microfluidic platform to report enzyme molecular weight, amount, and activity (including k(cat) and K(m)) from dilute protein mixtures. Calf intestinal alkaline phosphatase (CIP) is examined in detail as a model enzyme system, and the method is also demonstrated for horseradish peroxidase (HRP). The 40 min assay has a detection limit of 5 zmol ( approximately 3 000 molecules) of CIP. Two-step pore-limit electrophoresis with enzyme assay (PLENZ) is conducted in a single, straight microchannel housing a polyacrylamide (PA) pore-size gradient gel. In the first step, pore limit electrophoresis (PLE) sizes and pseudoimmobilizes resolved proteins. In the second step, electrophoresis transports both charged and neutral substrates into the PLE channel to the entrapped proteins. Arrival of substrate at the resolved enzyme band generates fluorescent product that reveals enzyme molecular weight against a fluorescent protein ladder. Additionally, the PLENZ zymography assay reports the kinetic properties of CIP in a fully quantitative manner. In contrast to covalent enzyme immobilization, physical pseudoimmobilization of CIP in the PA gel does not significantly reduce its maximum substrate turnover rate. However, an 11-fold increase in the Michaelis constant (over the free solution value) is observed, consistent with diffusional limitations on substrate access to the enzyme active site. PLENZ offers a robust platform for rapid and multiplexed functional analysis of heterogeneous protein samples in drug discovery, clinical diagnostics, and biocatalyst engineering.

  13. Soil microbial abundances and enzyme activities in different rhizospheres in an integrated vertical flow constructed wetland

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Ying; Jiang, Yueping; Jiang, Qinsu; Min, Hang; Fan, Haitian; Zeng, Qiang; Chang, Jie [College of Life Sciences, Zhejiang University, Hangzhou (China); Zhang, Chongbang [School of Life Sciences, Taizhou University, Linhai (China); Yue, Chunlei [Zhejiang Forestry Academy, Hangzhou (China)

    2011-03-15

    Rhizosphere microorganism is an important bio-component for wastewater treatment in constructed wetlands (CWs). Microbial abundance and enzyme activities in the rhizospheres of nine plant species were investigated in an integrated vertical-flow CW. The abundance of denitrifiers, as well as urease, acid, and alkaline phosphatase activities were positively correlated to plant root biomass. The abundance of bacteria, fungi, actinomycetes, ammonifiers, denitrifiers, and phosphorus decomposers, related to nutrient removal efficiencies in CWs, greatly varied among rhizospheres of different plant species (p < 0.05). Significant differences in rhizosphere enzyme activity among plant species were also observed (p < 0.05), with the exception of catalase activity. The principal component analysis using the data of microbial abundance and enzyme activity showed that Miscanthus floridulus, Acorus calamus, and Reineckia carnea were candidates to be used in CWs to effectively remove nitrogen and phosphorus from wastewater. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

    Science.gov (United States)

    Li, Mei-Hui

    2016-08-01

    The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities. Copyright © 2016. Published by Elsevier Inc.

  15. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    Science.gov (United States)

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  16. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    Energy Technology Data Exchange (ETDEWEB)

    Semsang, Nuananong, E-mail: nsemsang@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, LiangDeng [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

    2013-07-15

    Highlights: ► Ion beam bombarded rice seeds in vacuum. ► Studied seed survival from the ion bombardment. ► Determined various antioxidant enzyme activities and lipid peroxidation level. ► Discussed vacuum, ion species and ion energy effects. ► Attributed the changes to free radical formation due to ion bombardment. -- Abstract: Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29–60 keV and ion fluences of 1 × 10{sup 16} ions cm{sup −2}. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  17. Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qiang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); The First Affiliated Hospital of Xiamen University, Xiamen (China); Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); Yu, Chundong, E-mail: cdyu@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China)

    2011-06-17

    Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

  18. changes in activities of enzymes of glutamate metabolism in rat ...

    African Journals Online (AJOL)

    F , Cote, LJ , Ginsburg, S , Lawrence. G.D., Naini. P.. and Sano. M. (1990). Studies on new centrally active reversibie acety1cholinesterase inhibitors. Neurochem. Res. 15: 587-59. Barchas, J. D., Akii, H., Eiliot, G.R., Hoiman. B. and Watson. S.J (1978). Behavioral neurochemistry: Neuroreguiarors and behavioral states.

  19. Activity of Redox Enzymes in the Thallus of Anthoceros natalensis.

    Science.gov (United States)

    Chasov, A V; Beckett, R P; Minibayeva, F V

    2015-09-01

    Anthocerotophyta (hornworts) belong to a group of ancient nonvascular plants and originate from a common ancestor with contemporary vascular plants. Hornworts represent a unique model for investigating mechanisms of formation of stress resistance in higher plants due to their high tolerance to the action of adverse environmental factors. In this work, we demonstrate that the thallus of Anthoceros natalensis exhibits high redox activity changing under stress. Dehydration of the thallus is accompanied by the decrease in activities of intracellular peroxidases, DOPA-peroxidases, and tyrosinases, while catalase activity increases. Subsequent rehydration results in the increase in peroxidase and catalase activities. Kinetic features of peroxidases and tyrosinases were characterized as well as the peroxidase isoenzyme composition of different fractions of the hornwort cell wall proteins. It was shown that the hornwort peroxidases are functionally similar to peroxidases of higher vascular plants including their ability to form superoxide anion-radical. The biochemical mechanism was elucidated, supporting the possible participation of peroxidases in the formation of reactive oxygen species (ROS) via substrate-substrate interactions in the hornwort thallus. It has been suggested that the ROS formation by peroxidases is an evolutionarily ancient process that emerged as a protective mechanism for enhancing adaptive responses of higher land plants and their adaptation to changing environmental conditions and successful colonization of various ecological niches.

  20. activity of enzyme trypsin immobilized onto macroporous poly(epoxy

    African Journals Online (AJOL)

    dell

    storage conditions. According to Goradia et al. (2005) and Maurich et al. (2008) immobilization prevents autolysis thus making the immobilized trypsin relatively more stable than the free trypsin under the same storage conditions. The immobilized trypsin could be reused up to seven times without significant loss of activity.

  1. Enzyme Activity and Biomolecule Templating at Liquid and Solid Interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Harvey W. Blanch

    2004-12-01

    There are two main components of this research program. The first involves studies of the adsorption and catalytic activity of proteins at fluid-fluid and fluid-solid interfaces; the second employs biological macromolecules as templates at the solid-liquid interface for controlled crystallization of inorganic materials, to provide materials with specific functionality.

  2. Revealing hidden effect of earthworm on C distribution and enzyme activity

    Science.gov (United States)

    Razavi, Bahar S.; Hoang, Duyen; Kuzyakov, Yakov

    2017-04-01

    Despite its importance for terrestrial nutrient and carbon cycling, the spatial organization and localization of microbial activity in soil and in biopores is poorly understood. We hypothesized that biopores created by earthworm play a critical role in reducing the gap of SOM input and microbial activities between topsoil and subsoil. Accordingly, Carbon (C) allocation by earthworms was related to enzyme distribution along soil profile. For the first time we visualized spatial distribution of enzyme activities (β-glucosidase, chitinase and acid phosphatase) and C allocation (by 14C imaging) in earthworm biopores in topsoil and subsoil. Soil zymography (an in situ method for the analysis of the two-dimensional distribution of enzyme activity in soil) was accompanied with 14C imaging (a method that enables to trace distribution of litter and C in soil profile) to visualize change of enzyme activities along with SOM incorporation by earthworms from topsoil to subsoil. Experiment was set up acquiring rhizoboxes (9×1×50 cm) filled up with fresh soil and 3 earthworms (L. terrestris), which were then layered with 14C-labeled plant-litter of 0.3 MBq on the soil surface. 14C imaging and zymography have been carried out after one month. Activities of all enzymes regardless of their nutrient involvement (C, N, P) were higher in the biopores than in bulk soil, but the differences were larger in topsoil compared to subsoil. Among three enzymes, Phosphatase activity was 4-times higher in the biopore than in the bulk soil. Phosphatase activity was closely associated with edge of burrows and correlate positively with 14C activity. These results emphasized especial contribution of hotspheres such as biopores to C allocation in subsoil - which is limited in C input and nutrients - and in stimulation of microbial and enzymatic activity by input of organic residues, e.g. by earthworms. In conclusion, biopore increased enzymatic mobilization of nutrients (e.g. P) inducing allocation

  3. Heterogeneity of hydrolytic enzyme activities under drought: imaging and quantitative analysis

    Science.gov (United States)

    Sanaullah, Muhammad; Razavi, Bahar S.; Kuzyakov, Yakov

    2015-04-01

    The zymography-based "snap-shoot" of enzyme activities in the rhizosphere is challenging to detect the in situ microbial response to global climate change. We developed in situ soil zymography and used it for identification and localization of hotspots of β-glucosidase activity in the rhizosphere of maize under drought stress (30% of field capacity). The zymographic signals were especially high at root tips and were much stronger for activity of β-glucosidase under drought as compared with optimal moisture (70% of field capacity). This distribution of enzyme activity was confirmed by fluorogenically labelled substrates applied directly to the root exudates. The activity of β-glucosidase in root exudates (produced by root and microorganism associated on the root surface), sampled within 1 hour after zymography was significantly higher by drought stressed plants as compared with optimal moisture. In contrast, the β-glucosidase activity in destructively sampled rhizosphere soil was lower under drought stress compared with optimal moisture. Furthermore, drought stress did not affected β-glucosidase activity in bulk soil, away from rhizosphere. Consequently, we conclude that higher release of mucilage by roots und drought stimulated β-glucosidase activity in the rhizosphere. Thus, the zymography revealed plant-mediated mechanisms accelerating β-glucosidase activity under drought at the root-soil interface. So, coupling of zymography and enzyme assays in the rhizosphere and non-rhizosphere soil enables precise mapping the changes in two-dimensional distribution of enzyme activities due to climate change within dynamic soil interfaces.

  4. Characterization of AmiBA2446, a novel bacteriolytic enzyme active against Bacillus species.

    Science.gov (United States)

    Mehta, Krunal K; Paskaleva, Elena E; Azizi-Ghannad, Saba; Ley, Daniel J; Page, Martin A; Dordick, Jonathan S; Kane, Ravi S

    2013-10-01

    There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

  5. Redox enzyme-mimicking activities of CeO2nanostructures: Intrinsic influence of exposed facets.

    Science.gov (United States)

    Yang, Yushi; Mao, Zhou; Huang, Wenjie; Liu, Lihua; Li, Junli; Li, Jialiang; Wu, Qingzhi

    2016-10-17

    CeO 2 nanoparticles (NPs) have been well demonstrated as an antioxidant in protecting against oxidative stress-induced cellular damages and a potential therapeutic agent for various diseases thanks to their redox enzyme-mimicking activities. The Ce 3+ /Ce 4+ ratio and oxygen vacancies on the surface have been considered as the major originations responsible for the redox enzyme-mimicking activities of CeO 2 NPs. Herein, CeO 2 nanostructures (nanocubes and nanorods) exposed different facets were synthesized via a facile hydrothermal method. The characterizations by X-ray photoelectron spectroscopy, Raman spectroscopy, and UV-Vis spectroscopy show that the Ce 3+ /Ce 4+ ratio and oxygen vacancy content on the surfaces of as-synthesized CeO 2 nanostructures are nearly at the same levels. Meanwhile, the enzymatic activity measurements indicate that the redox enzyme-mimicking activities of as-synthesized CeO 2 nanostructures are greatly dependent on their exposed facets. CeO 2 nanocubes with exposed {100} facets exhibit a higher peroxidase but lower superoxide dismutase activity than those of the CeO 2 nanorods with exposed {110} facets. Our results provide new insights into the redox enzyme-mimicking activities of CeO 2 nanostructures, as well as the design and synthesis of inorganic nanomaterials-based artificial enzymes.

  6. [The restoration of the enzyme activity of chernozem soil after gamma-irradiation].

    Science.gov (United States)

    Denisova, T V; Kazeev, K Sh

    2006-01-01

    The Influence of gamma-radiation by dozes 1, 5, 10 and 20 kGy on enzyme activity of ordinary chemozem were studied. Dynamics of the restoration of the enzyme activity after the influence of gamma-radiation in model experiments in 3, 30, 90 and 180 days was investigated. The doze 1 kGy did no statistically significant influence on the investigated enzymes. Dehydrogenase is more radiosensitive enzyme than catalase. Values of the saccharase activity differed a significant variation so in most cases it has not been registered statistically significant changes. In 90-180 days of the incubation enzymes activity was restored up to control values. Dehydrogenase activity in 180 days in variants with dozes 10 and 20 kGy was restored up to a level of the control, over variants with dozes 1 and 5 kGy--is higher than the control over 78% and 23% accordingly. Saccharase activity in 180 days after the influence of gamma-radiation with a doze 20 kGy was on 61% lower than the control.

  7. Effect of bleaching on mercury release from amalgam fillings and antioxidant enzyme activities: a pilot study.

    Science.gov (United States)

    Cakir, Filiz Yalcin; Ergin, Esra; Gurgan, Sevil; Sabuncuoglu, Suna; Arpa, Cigdem Sahin; Tokgoz, İlknur; Ozgunes, Hilal; Kiremitci, Arlin

    2015-01-01

    The aim of this pilot clinical study was to determine the mercury release from amalgam fillings and antioxidant enzyme activities (Superoxide Dismutase [SOD] and Catalase[CAT] ) in body fluids after exposure to two different vital tooth bleaching systems. Twenty eight subjects with an average age of 25.6 years (18-41) having at least two but not more than four Class II amalgam fillings on each quadrant arch in the mouth participated in the study. Baseline concentrations of mercury levels in whole blood, urine, and saliva were measured by a Vapor Generation Accessory connected to an Atomic Absorption Spectrometer. Erythrocyte enzymes, SOD, and CAT activities in blood were determined kinetically. Subjects were randomly assigned to two groups of 14 volunteers. Group 1 was treated with an at-home bleaching system (Opalescence PF 35% Carbamide Peroxide, Ultradent), and Group 2 was treated with a chemically activated office bleaching system (Opalescence Xtra Boost 38% Hydrogen Peroxide, Ultradent) according to the manufacturer's recommendations. Twenty-four hours after bleaching treatments, concentrations of mercury and enzymes were remeasured. There were no significant differences on mercury levels in blood, urine, and saliva before and after bleaching treatments (p > 0.05). No differences were also found in the level of antioxidant enzyme activities (SOD and CAT) before and after treatments (p > 0.05). Mercury release did not affect the enzyme activities (p > 0.05). Bleaching treatments either office or home did not affect the amount of mercury released from amalgam fillings in blood, urine, and saliva and the antioxidant-enzyme activities in blood. Bleaching treatments with the systems tested in this pilot study have no deleterious effect on the mercury release from amalgam fillings and antioxidant enzymes in body fluids. © 2014 Wiley Periodicals, Inc.

  8. FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation.

    Directory of Open Access Journals (Sweden)

    Teresa Koller

    Full Text Available Signaling initiation by receptor-like kinases (RLKs at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP receptor FLS2 builds signaling complexes with BAK1 (SERK3. We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs of the significantly different receptors FLS2, EFR (MAMP receptors, PEPR1 (damage-associated molecular pattern (DAMP receptor, and BRI1 (hormone receptor. Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants

  9. Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities

    International Nuclear Information System (INIS)

    Cang Long; Zhou Dongmei; Wang Quanying; Wu Danya

    2009-01-01

    There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm -1 of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P < 0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

  10. Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities

    Energy Technology Data Exchange (ETDEWEB)

    Cang Long [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China); Zhou Dongmei, E-mail: dmzhou@issas.ac.cn [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Wang Quanying; Wu Danya [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China)

    2009-12-30

    There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm{sup -1} of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P < 0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

  11. Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.

    Science.gov (United States)

    Herz, Daniel B; Aitken, Karen; Bagli, Darius J

    2003-11-01

    Bladders clinically subjected to excessive pressure or distention demonstrate an altered extracellular matrix (ECM) composition. We determined how an altered collagen substratum might affect bladder smooth muscle cell (bSMC) growth in vitro and probed the mechanism of this response. Primary culture rat bSMCs were seeded onto culture plates pre-coated with normal type I collagen (NC) or heat denatured type I collagen (DNC) under standard culture conditions. In separate experiments bSMCs from the 2 substrates were enzymatically released and changed to growth on normal collagen (NC-->NC or DNC-->NC) or denatured collagen (DNC-->DNC or NC-->DNC). At 24 hours proliferation was assessed by 3H-thymidine incorporation. Statistical significance in triplicate wells was determined by ANOVA. The proliferation of bSMCs on DNC was 5-fold greater than on NC (p DNC-->DNC) showed 2-fold further augmentation in proliferation (p DNC-->NC) (p NC) generated a 33% decrease in the already low proliferation rate (p DNC) (p DNC. However, mitogenicity is only partially reversible by re-introducing NC. These results demonstrate striking bSMC responsiveness to ECM conformation. Signaling through the extracellular regulated kinase mitogen activated protein kinase pathway supports bSMC-ECM interaction. We speculate that remodeling the ECM in vivo may regulate bSMC growth.

  12. Fractones: extracellular matrix niche controlling stem cell fate and growth factor activity in the brain in health and disease.

    Science.gov (United States)

    Mercier, Frederic

    2016-12-01

    The stem cell niche refers to a specific microenvironment where stem cells proliferate and differentiate to produce new specialized cells throughout an organism's adulthood. Growth factors are crucial signaling molecules that diffuse through the extracellular space, reach the stem cell niche, and ultimately promote stem cell proliferation and differentiation. However, it is not well known how multiple growth factors, often with antagonistic activities, work together in the stem cell niche to select target stem cell populations and determine stem cell fate. There is accumulating evidence suggesting that extracellular matrix (ECM) molecules play an important role in promoting growth factor access and activity in the stem cell niche. In the adult brain neurogenic zone, where neural stem cells (NSCs) reside, there exist specialized ECM structures, which we have named fractones. The processes of NSC allow them to come into contact with fractones and interact with its individual components, which include heparan sulfate proteoglycans (HSPGs) and laminins. We have demonstrated that fractone-associated HSPGs bind growth factors and regulate NSC proliferation in the neurogenic zone. Moreover, emerging results show that fractones are structurally altered in animal models with autism and adult hydrocephalus, as demonstrated by changes in fractone size, quantity, or HSPG content. Interestingly, ECM structures similar to fractones have been found throughout β-amyloid plaques in the brain of patients with Alzheimer's disease. Pathological fractones may cause imbalances in growth factor activity and impair neurogenesis, leading to inflammation and disorder. Generally speaking, these stem cell niche structures play a potentially vital role in controlling growth factor activity during both health and disease.

  13. Is there any role of prolidase enzyme activity in the etiology of preeclampsia?

    Science.gov (United States)

    Pehlivan, Mustafa; Ozün Ozbay, Pelin; Temur, Muzaffer; Yılmaz, Ozgur; Verit, Fatma Ferda; Aksoy, Nurten; Korkmazer, Engin; Üstünyurt, Emin

    2017-05-01

    To evaluate a relationship between preeclampsia and prolidase enzyme activity. A prospective cohort study of 41 pregnant women diagnosed with preeclampsia and 31 healthy pregnant women as control group was selected at Harran University Hospital Department of Obstetrics and Gynecology. The prolidase enzyme activity was analyzed in maternal and umbilical cord plasma, amniotic fluid and placental and umbilical cord tissues by Chinard method in addition to maternal serum levels of lactate dehydrogenase (LDH), serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT). A significant relationship was found between plasma prolidase activity (635 ± 83 U/L) (p  = 0.007), umbilical cord plasma prolidase activity (610 ± 90 U/L) (p = 0.013), amniotic fluid prolidase activity (558 ± 100 U/L) (p  = 0.001), umbilical cord tissue prolidase activity (4248 ± 1675 U/gr protein) (p  = 0.013) and placental tissue prolidase activity (2116 ± 601 U/gr protein) (p  = 0.001) in preeclamptic group when compared to healthy pregnant women. There is a strong correlation between prolidase enzyme activity and preeclampsia. Prolidase enzyme activity may play a role in preeclampsia.

  14. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    Full Text Available Three hundred one-day-old male broiler chickens (Ross-308 were fed corn-soybean basal diets containing non-starch polysaccharide (NSP enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI and average daily gain (ADG were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05. Feed-to-gain ratio (FGR was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05. Apparent digestibility of crude protein (ADCP was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05. Cholecystokinin (CCK level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05, but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05, respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05. However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05. The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05. Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  15. Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus.

    Science.gov (United States)

    Liu, Haitian; Wang, Shifeng; Cai, Yan; Guo, Xiaohui; Cao, Zhenjie; Zhang, Yongzheng; Liu, Shubin; Yuan, Wei; Zhu, Weiwei; Zheng, Yu; Xie, Zhenyu; Guo, Weiliang; Zhou, Yongcan

    2017-01-01

    The probiotic properties of Bacillus subtilis HAINUP40 isolated from the aquatic environment, and the effects of dietary administration of B. subtilis HAINUP40 on the growth performance, intestinal probiotic recovery, digestive enzyme activities, innate immunity and disease resistance of tilapia (Oreochromis niloticus) were evaluated. The probiotic properties investigated include tolerance to simulated gastrointestinal stress, auto-aggregation, cell surface hydrophobicity and extracellular enzyme production. The cell number of B. subtilis changed little after 4 h in simulated gastric fluid at pH = 2.0, 3.0, 4.0 and simulated intestinal fluid at pH = 6.8.B.subtilis HAINUP40 revealed strong auto-aggregation property (34.6-87.0%) after 24 h incubation period. It exhibited significant cell surface hydrophobicity in xylene (28.8%) and chloroform (41.3%) and produced extracellular proteases and amylase. After tilapia (mean weight = 95 ± 8 g) were fed with a diet containing 10 8  cfu/g B. subtilis HAINUP40, their final body weight, percent weight gain (PWG), specific growth rate (SGR), total antioxidant capacity (T-AOC) and serum superoxide dismutase (SOD) increased significantly (p subtilis HAINUP40 can effectively enhances the growth performance, immune response, and disease resistance of Nile tilapia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Studies on the effects of radiation on enzyme activity and chromosome in mammals (Mus musuculus)

    International Nuclear Information System (INIS)

    Kim, J.B.; Lee, K.S.; Kim, Y.J.

    1982-01-01

    From the results of many researches in radiation biology, it is well known that the radiation induces gene mutation, aberration of chromosome which is a carrier of genes and the increase or decrease of enzyme activities in living organisms. However, the frequency of chromosomal aberration or the degree of enzyme activities according to the animal's age when they are irradiated with radiation and time pass after irradiation are known a little if any. From these viewpoints, the research on the frequencies of chromosomal aberrations in bone marrow cells and the degree of activities of glucose-6-phosphate dehydrogenase in liver, kidney and brain, and isocitrate dehydrogense in kidney and brain of mouse has been carried out according to the mice age when they are irradiated with 200 rad of whole body irradiation. The chromosomes and enzyme activities were observed at 24 hours, 48 hours and 4 days to 90 days after irradiation. (Author)

  17. Increased activities of mitochondrial enzymes in white adipose tissue in trained rats

    DEFF Research Database (Denmark)

    Stallknecht, B; Vinten, J; Ploug, T

    1991-01-01

    of 8-12 rats were swim trained for 10 wk or served as either sedentary, sham swim-trained, or cold-stressed controls. White adipose tissue was removed, and the activities of the respiratory chain enzyme cytochrome-c oxidase (CCO) and of the enzyme malate dehydrogenase (MDH), which participates...... 0.05). In female rats the CCO activity expressed per milligram protein was increased 4.5-fold in the trained compared with the sedentary control rats (P less than 0.01). Neither cold stress nor sham swim training increased CCO or MDH activities in white adipose tissue (P greater than 0...

  18. A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolase...

  19. Altered Activities of Antioxidant Enzymes in Patients with Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Lucie Vávrová

    2013-02-01

    Full Text Available Objective: In the pathogenesis of the metabolic syndrome (MetS, an increase of oxidative stress could play an important role which is closely linked with insulin resistance, endothelial dysfunction, and chronic inflammation. The aim of our study was to assess several parameters of the antioxidant status in MetS. Methods: 40 subjects with MetS and 40 age- and sex-matched volunteers without MetS were examined for activities of superoxide dismutase (CuZnSOD, catalase (CAT, glutathione peroxidase 1 (GPX1, glutathione reductase (GR, paraoxonase1 (PON1, concentrations of reduced glutathione (GSH, and conjugated dienes in low-density lipoprotein (CD-LDL. Results: Subjects with MetS had higher activities of CuZnSOD (p Conclusions: Our results implicated an increased oxidative stress in MetS and a decreased antioxidative defense that correlated with some laboratory (triglycerides, high-density lipoprotein cholesterol (HDL-C and clinical (waist circumference, blood pressure components of MetS.

  20. Soil microbial communities and enzyme activities in sea-buckthorn (Hippophae rhamnoides plantation at different ages.

    Directory of Open Access Journals (Sweden)

    Miao Yang

    Full Text Available The aim of this study was to assess the impact of forest age and season on the soil microbial community and enzyme activities in sea-buckthorn plantation system and to determine the relative contributions to soil microbial properties. Soil sampling was carried out in the dry season (April and wet season (September in four areas, including: abandoned farmland (NH, an 8-year- old plantation (young plantation, 8Y, a 13-year-old plantation (middle-aged plantation, 13Y, and an 18-year-old plantation (mature plantation, 18Y. The results showed that forest age and season have a significant effect on soil microbial community structure and enzyme activities. The total, bacterial, fungal, Gram-negative (G+, and Gram-positive (G- PLFAs increased gradually with forest age, with the highest values detected in 18Y. All the detected enzyme activities showed the trend as a consequence of forest age. The microbial PLFAs and soil enzyme activities were higher in the wet season than the dry season. However, there were no significant interactions between forest age and season. A Correlation analysis suggested that soil microbial communities and enzyme activities were significantly and positively correlated with pH, total nitrogen (TN and available phosphorus (AP. Season had a stronger influence on soil microbial communities than forest age. In general, sea-buckthorn plantations establishment might be a potential tool for maintaining and increasing soil fertility in arid and semi-arid regions.

  1. Enhanced active extracellular polysaccharide production from Ganoderma formosanum using computational modeling.

    Science.gov (United States)

    Hsu, Kai-Di; Wu, Shu-Pei; Lin, Shin-Ping; Lum, Chi-Chin; Cheng, Kuan-Chen

    2017-10-01

    Extracellular polysaccharide (EPS) is one of the major bioactive ingredients contributing to the health benefits of Ganoderma spp. In this study, response surface methodology was applied to determine the optimal culture conditions for EPS production of Ganoderma formosanum. The optimum medium composition was found to be at initial pH 5.3, 49.2 g/L of glucose, and 4.9 g/L of yeast extract by implementing a three-factor-three-level Box-Behnken design. Under this condition, the predicted yield of EPS was up to 830.2 mg/L, which was 1.4-fold higher than the one from basic medium (604.5 mg/L). Furthermore, validating the experimental value of EPS production depicted a high correlation (100.4%) with the computational prediction response model. In addition, the percentage of β-glucan, a well-recognized bioactive polysaccharide, in EPS was 53±5.5%, which was higher than that from Ganoderma lucidum in a previous study. Moreover, results of monosaccharide composition analysis indicated that glucose was the major component of G. formosanum EPS, supporting a high β-glucan percentage in EPS. Taken together, this is the first study to investigate the influence of medium composition for G. formosanum EPS production as well as its β-glucan composition. Copyright © 2017. Published by Elsevier B.V.

  2. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  3. Effects of silver nanoparticles on soil enzyme activities with and without added organic matter.

    Science.gov (United States)

    Peyrot, Caroline; Wilkinson, Kevin J; Desrosiers, Mélanie; Sauvé, Sébastien

    2014-01-01

    The effects of silver nanoparticles (AgNPs) on terrestrial ecosystems need to be better understood and assessed. Cationic silver (Ag+) has well-documented toxicity against bacteria, but it is not clear what will be the effect of nanoscale Ag. In the present study, the potential effects of AgNPs were investigated in soils by measuring activity of the enzymes phosphomonoesterase, arylsulfatase, β-D-glucosidase, and leucine-aminopeptidase. The toxicity of AgNPs was compared with that of ionic Ag, and the ameliorating effects of soil organic matter were evaluated. To this end, 2 soils with different organic matter contents were artificially contaminated with either AgNPs or Ag-acetate at equivalent total Ag concentrations. In general, enzyme activities were inhibited as a function of the Ag concentration in the soil. In the AgNP exposures, only a small fraction of the AgNP was actually truly dissolved (found in the soil enzymes. The addition of organic matter to the soils appeared to enhance enzyme activities; however, the mechanism of organic matter action is not clear given that dissolved Ag concentrations were similar in both the organic-matter–amended and unamended soils. The present study shows that the AgNP produces significant negative effects on the soil enzyme activities tested. The Ag chemical speciation measurements suggested that the AgNP caused greater toxic effects to the soil enzymes at the low Ag concentrations. For the larger concentrations of total soil Ag, causes of the negative effects on enzyme activities are less obvious but suggest that colloidal forms of Ag play a role.

  4. CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum.

    Science.gov (United States)

    Kersey, Caleb M; Agyemang, Paul A; Dumenyo, C Korsi

    2012-01-01

    Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  5. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

    DEFF Research Database (Denmark)

    Ørsted, Michael; Roslev, Peter

    2015-01-01

    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme