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Sample records for highly conserved enzymes

  1. Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

    Directory of Open Access Journals (Sweden)

    Ranyee A Chiang

    2008-08-01

    Full Text Available The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized

  2. Evolutionarily conserved substrate substructures for automated annotation of enzyme superfamilies.

    Science.gov (United States)

    Chiang, Ranyee A; Sali, Andrej; Babbitt, Patricia C

    2008-08-01

    The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized and uncharacterized

  3. Energy conservation and maximal entropy production in enzyme reactions.

    Science.gov (United States)

    Dobovišek, Andrej; Vitas, Marko; Brumen, Milan; Fajmut, Aleš

    2017-08-01

    A procedure for maximization of the density of entropy production in a single stationary two-step enzyme reaction is developed. Under the constraints of mass conservation, fixed equilibrium constant of a reaction and fixed products of forward and backward enzyme rate constants the existence of maximum in the density of entropy production is demonstrated. In the state with maximal density of entropy production the optimal enzyme rate constants, the stationary concentrations of the substrate and the product, the stationary product yield as well as the stationary reaction flux are calculated. The test, whether these calculated values of the reaction parameters are consistent with their corresponding measured values, is performed for the enzyme Glucose Isomerase. It is found that calculated and measured rate constants agree within an order of magnitude, whereas the calculated reaction flux and the product yield differ from their corresponding measured values for less than 20 % and 5 %, respectively. This indicates that the enzyme Glucose Isomerase, considered in a non-equilibrium stationary state, as found in experiments using the continuous stirred tank reactors, possibly operates close to the state with the maximum in the density of entropy production. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

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    Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

    2010-12-01

    We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

  5. 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

  6. Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    Directory of Open Access Journals (Sweden)

    Janine Maimanakos

    2016-08-01

    Full Text Available Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76 are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99% of the (R-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

  7. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  8. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

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    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  9. 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 ...

  10. The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

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    Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

    2006-01-01

    Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

  11. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Jørgensen, Henning

    2013-01-01

    the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

  12. Modification of enzymes by use of high-pressure homogenization.

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    Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

    2018-07-01

    High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Constrained dansyl derivatives reveal bacterial specificity of highly conserved thymidylate synthases.

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    Calò, Sanuele; Tondi, Donatella; Ferrari, Stefania; Venturelli, Alberto; Ghelli, Stefano; Costi, Maria Paola

    2008-03-25

    The elucidation of the structural/functional specificities of highly conserved enzymes remains a challenging area of investigation, and enzymes involved in cellular replication are important targets for functional studies and drug discovery. Thymidylate synthase (TS, ThyA) governs the synthesis of thymidylate for use in DNA synthesis. The present study focused on Lactobacillus casei TS (LcTS) and Escherichia coli TS (EcTS), which exhibit 50 % sequence identity and strong folding similarity. We have successfully designed and validated a chemical model in which linear, but not constrained, dansyl derivatives specifically complement the LcTS active site. Conversely, chemically constrained dansyl derivatives showed up to 1000-fold improved affinity for EcTS relative to the inhibitory activity of linear derivatives. This study demonstrates that the accurate design of small ligands can uncover functional features of highly conserved enzymes.

  14. Readings in Wildlife and Fish Conservation, High School Conservation Curriculum Project.

    Science.gov (United States)

    Ensminger, Jack

    This publication is a tentative edition of readings on Wildlife and Fish Conservation in Louisiana, and as such it forms part of one of the four units of study designed for an experimental high school course, the "High School Conservation Curriculum Project." The other three units are concerned with Forest Conervation, Soil and Water…

  15. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  16. Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

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    Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

    2015-01-01

    Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

  17. Highly conserved small subunit residues influence rubisco large subunit catalysis.

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    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  18. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  19. Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

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    Patrick E F O'Rourke

    Full Text Available 4-Amino-4-deoxychorismate lyase (PabC catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5'-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp(2 to sp(3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of

  20. High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

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    Akazawa, Shin-Ichi; Tokuyama, Haruka; Sato, Shunsuke; Watanabe, Toshinori; Shida, Yosuke; Ogasawara, Wataru

    2018-02-01

    Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1

    Science.gov (United States)

    Moghe, Gaurav D.; Last, Robert L.

    2015-01-01

    Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks. PMID:26276843

  2. Tilapia and human CLIC2 structures are highly conserved.

    Science.gov (United States)

    Zeng, Jiao; Li, Zhengjun; Lui, Eei Yin; Lam, Siew Hong; Swaminathan, Kunchithapadam

    2018-01-08

    Chloride intracellular channels (CLICs) exist in soluble and membrane bound forms. We have determined the crystal structure of soluble Clic2 from the euryhaline teleost fish Oreochromis mossambicus. Structural comparison of tilapia and human CLIC2 with other CLICs shows that these proteins are highly conserved. We have also compared the expression levels of clic2 in selected osmoregulatory organs of tilapia, acclimated to freshwater, seawater and hypersaline water. Structural conservation of vertebrate CLICs implies that they might play conserved roles. Also, tissue-specific responsiveness of clic2 suggests that it might be involved in iono-osmoregulation under extreme conditions in tilapia. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    Science.gov (United States)

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  4. Conservation

    NARCIS (Netherlands)

    Noteboom, H.P.

    1985-01-01

    The IUCN/WWF Plants Conservation Programme 1984 — 1985. World Wildlife Fund chose plants to be the subject of their fund-raising campaign in the period 1984 — 1985. The objectives were to: 1. Use information techniques to achieve the conservation objectives of the Plants Programme – to save plants;

  5. Conservation.

    Science.gov (United States)

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  6. A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species.

    Directory of Open Access Journals (Sweden)

    Adnane Boualem

    Full Text Available Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of

  7. Structure of Human cGAS Reveals a Conserved Family of Second-Messenger Enzymes in Innate Immunity

    Directory of Open Access Journals (Sweden)

    Philip J. Kranzusch

    2013-05-01

    Full Text Available Innate immune recognition of foreign nucleic acids induces protective interferon responses. Detection of cytosolic DNA triggers downstream immune signaling through activation of cyclic GMP-AMP synthase (cGAS. We report here the crystal structure of human cGAS, revealing an unanticipated zinc-ribbon DNA-binding domain appended to a core enzymatic nucleotidyltransferase scaffold. The catalytic core of cGAS is structurally homologous to the RNA-sensing enzyme, 2′-5′ oligo-adenylate synthase (OAS, and divergent C-terminal domains account for specific ligand-activation requirements of each enzyme. We show that the cGAS zinc ribbon is essential for STING-dependent induction of the interferon response and that conserved amino acids displayed within the intervening loops are required for efficient cytosolic DNA recognition. These results demonstrate that cGAS and OAS define a family of innate immunity sensors and that structural divergence from a core nucleotidyltransferase enables second-messenger responses to distinct foreign nucleic acids.

  8. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  9. Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement

    International Nuclear Information System (INIS)

    Broderick, T.P.; Schaff, D.A.; Bertino, A.M.; Dush, M.K.; Tischfield, J.A.; Stambrook, P.J.

    1987-01-01

    The functional human adenine phosphoribosyltransferase (APRT) gene is <2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other housekeeping genes, lacks TATA and CCAAT boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function

  10. Hierarchically Nanoporous Bioactive Glasses for High Efficiency Immobilization of Enzymes

    DEFF Research Database (Denmark)

    He, W.; Min, D.D.; Zhang, X.D.

    2014-01-01

    Bioactive glasses with hierarchical nanoporosity and structures have been heavily involved in immobilization of enzymes. Because of meticulous design and ingenious hierarchical nanostructuration of porosities from yeast cell biotemplates, hierarchically nanostructured porous bioactive glasses can...... and products of catalytic reactions can freely diffuse through open mesopores (2–40 nm). The formation mechanism of hierarchically structured porous bioactive glasses, the immobilization mechanism of enzyme and the catalysis mechanism of immobilized enzyme are then discussed. The novel nanostructure...

  11. Evolutionary growth process of highly conserved sequences in vertebrate genomes.

    Science.gov (United States)

    Ishibashi, Minaka; Noda, Akiko Ogura; Sakate, Ryuichi; Imanishi, Tadashi

    2012-08-01

    Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

  13. ABCE1 is a highly conserved RNA silencing suppressor.

    Directory of Open Access Journals (Sweden)

    Kairi Kärblane

    Full Text Available ATP-binding cassette sub-family E member 1 (ABCE1 is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

  14. High-resolution finite-difference algorithms for conservation laws

    International Nuclear Information System (INIS)

    Towers, J.D.

    1987-01-01

    A new class of Total Variation Decreasing (TVD) schemes for 2-dimensional scalar conservation laws is constructed using either flux-limited or slope-limited numerical fluxes. The schemes are proven to have formal second-order accuracy in regions where neither u/sub x/ nor y/sub y/ vanishes. A new class of high-resolution large-time-step TVD schemes is constructed by adding flux-limited correction terms to the first-order accurate large-time-step version of the Engquist-Osher scheme. The use of the transport-collapse operator in place of the exact solution operator for the construction of difference schemes is studied. The production of spurious extrema by difference schemes is studied. A simple condition guaranteeing the nonproduction of spurious extrema is derived. A sufficient class of entropy inequalities for a conservation law with a flux having a single inflection point is presented. Finite-difference schemes satisfying a discrete version of each entropy inequality are only first-order accurate

  15. Dynamic Epigenetic Control of Highly Conserved Noncoding Elements

    KAUST Repository

    Seridi, Loqmane

    2014-10-07

    Background Many noncoding genomic loci have remained constant over long evolutionary periods, suggesting that they are exposed to strong selective pressures. The molecular functions of these elements have been partially elucidated, but the fundamental reason for their extreme conservation is still unknown. Results To gain new insights into the extreme selection of highly conserved noncoding elements (HCNEs), we used a systematic analysis of multi-omic data to study the epigenetic regulation of such elements during the development of Drosophila melanogaster. At the sequence level, HCNEs are GC-rich and have a characteristic oligomeric composition. They have higher levels of stable nucleosome occupancy than their flanking regions, and lower levels of mononucleosomes and H3.3, suggesting that these regions reside in compact chromatin. Furthermore, these regions showed remarkable modulations in histone modification and the expression levels of adjacent genes during development. Although HCNEs are primarily initiated late in replication, about 10% were related to early replication origins. Finally, HCNEs showed strong enrichment within lamina-associated domains. Conclusion HCNEs have distinct and protective sequence properties, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. These observations indicate that such elements are likely to have essential cellular functions, and offer insights into their epigenetic properties.

  16. Dynamic Epigenetic Control of Highly Conserved Noncoding Elements

    KAUST Repository

    Seridi, Loqmane; Ryu, Tae Woo; Ravasi, Timothy

    2014-01-01

    Background Many noncoding genomic loci have remained constant over long evolutionary periods, suggesting that they are exposed to strong selective pressures. The molecular functions of these elements have been partially elucidated, but the fundamental reason for their extreme conservation is still unknown. Results To gain new insights into the extreme selection of highly conserved noncoding elements (HCNEs), we used a systematic analysis of multi-omic data to study the epigenetic regulation of such elements during the development of Drosophila melanogaster. At the sequence level, HCNEs are GC-rich and have a characteristic oligomeric composition. They have higher levels of stable nucleosome occupancy than their flanking regions, and lower levels of mononucleosomes and H3.3, suggesting that these regions reside in compact chromatin. Furthermore, these regions showed remarkable modulations in histone modification and the expression levels of adjacent genes during development. Although HCNEs are primarily initiated late in replication, about 10% were related to early replication origins. Finally, HCNEs showed strong enrichment within lamina-associated domains. Conclusion HCNEs have distinct and protective sequence properties, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. These observations indicate that such elements are likely to have essential cellular functions, and offer insights into their epigenetic properties.

  17. High cumulants of conserved charges and their statistical uncertainties

    Science.gov (United States)

    Li-Zhu, Chen; Ye-Yin, Zhao; Xue, Pan; Zhi-Ming, Li; Yuan-Fang, Wu

    2017-10-01

    We study the influence of measured high cumulants of conserved charges on their associated statistical uncertainties in relativistic heavy-ion collisions. With a given number of events, the measured cumulants randomly fluctuate with an approximately normal distribution, while the estimated statistical uncertainties are found to be correlated with corresponding values of the obtained cumulants. Generally, with a given number of events, the larger the cumulants we measure, the larger the statistical uncertainties that are estimated. The error-weighted averaged cumulants are dependent on statistics. Despite this effect, however, it is found that the three sigma rule of thumb is still applicable when the statistics are above one million. Supported by NSFC (11405088, 11521064, 11647093), Major State Basic Research Development Program of China (2014CB845402) and Ministry of Science and Technology (MoST) (2016YFE0104800)

  18. Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

    Science.gov (United States)

    Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2014-03-18

    Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. A highly conserved basidiomycete peptide synthetase produces a trimeric hydroxamate siderophore.

    Science.gov (United States)

    Brandenburger, Eileen; Gressler, Markus; Leonhardt, Robin; Lackner, Gerald; Habel, Andreas; Hertweck, Christian; Brock, Matthias; Hoffmeister, Dirk

    2017-08-25

    The model white-rot basidiomycete Ceriporiopsis ( Gelatoporia ) subvermispora B encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet uncharacterized fungal type VI siderophore synthetase family which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module and two thiolation/condensation (T-C) di-domain partial modules which, together, constitute an AT 1 C 1 T 2 C 2 T 3 C 3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as polyhistidine fusion in Aspergillus niger as soluble and active protein. N 5 -acetyl- N 5 -hydroxy-l-ornithine (l-AHO) and N 5 - cis -anhydromevalonyl- N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as substrates, as assessed in vitro using the substrate-dependent [ 32 P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo -CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as product in vitro , which was verified by LC-HRESIMS. Phylogenetic analyses suggest that type VI family siderophore synthetases are widespread in mushrooms and have evolved in a common ancestor of basidiomycetes. Importance : The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 supports further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the

  20. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

  1. Inhibition of raw starch digestion by one glucoamylase preparation from black Aspergillus at high enzyme concentration

    Energy Technology Data Exchange (ETDEWEB)

    Saka, B C; Veda, S

    1981-09-01

    Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60 deg. Celcius or above. (Refs. 11).

  2. Eucaryotic operon genes can define highly conserved syntenies

    Czech Academy of Sciences Publication Activity Database

    Trachtulec, Zdeněk

    2004-01-01

    Roč. 50, - (2004), s. 1-6 ISSN 0015-5500 R&D Projects: GA ČR GA204/01/0997; GA MŠk LN00A079 Institutional research plan: CEZ:AV0Z5052915 Keywords : eukaryotic operon * conserved synteny Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.507, year: 2004

  3. Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

    Directory of Open Access Journals (Sweden)

    Supaporn Kradtap Hartwell

    2012-01-01

    Full Text Available Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

  4. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

    International Nuclear Information System (INIS)

    Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

    2016-01-01

    A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-L-arginine ethyl ester to N_α-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

  5. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

    Energy Technology Data Exchange (ETDEWEB)

    Ruan, Guihua, E-mail: guihuaruan@hotmail.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China); Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Du, Fuyou, E-mail: dufu2005@126.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China)

    2016-04-22

    A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

  6. Energy conservation and pomeron loops in high energy evolution

    International Nuclear Information System (INIS)

    Gustafson, Goesta

    2007-01-01

    We present a formalism which modifies the Mueller Dipole Model such that it incorporates energy-momentum conservation as well as important colour suppressed effects in the cascade evolution. The formalism is implemented in a Monte Carlo simulation program, and the results are compared to inclusive data from HERA and the Tevatron. We here find a generally very good agreement between our model and the experimental data. (author)

  7. Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics

    Science.gov (United States)

    2015-01-01

    The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings. PMID:26704937

  8. Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits.

    Science.gov (United States)

    Choi, Su-Lim; Rha, Eugene; Lee, Sang Jun; Kim, Haseong; Kwon, Kilkoang; Jeong, Young-Su; Rhee, Young Ha; Song, Jae Jun; Kim, Hak-Sung; Lee, Seung-Goo

    2014-03-21

    Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

  9. Usherin expression is highly conserved in mouse and human tissues.

    Science.gov (United States)

    Pearsall, Nicole; Bhattacharya, Gautam; Wisecarver, Jim; Adams, Joe; Cosgrove, Dominic; Kimberling, William

    2002-12-01

    Usher syndrome is an autosomal recessive disease that results in varying degrees of hearing loss and retinitis pigmentosa. Three types of Usher syndrome (I, II, and III) have been identified clinically with Usher type II being the most common of the three types. Usher type II has been localized to three different chromosomes 1q41, 3p, and 5q, corresponding to Usher type 2A, 2B, and 2C respectively. Usherin is a basement membrane protein encoded by the USH2A gene. Expression of usherin has been localized in the basement membrane of several tissues, however it is not ubiquitous. Immunohistochemistry detected usherin in the following human tissues: retina, cochlea, small and large intestine, pancreas, bladder, prostate, esophagus, trachea, thymus, salivary glands, placenta, ovary, fallopian tube, uterus, and testis. Usherin was absent in many other tissues such as heart, lung, liver, kidney, and brain. This distribution is consistent with the usherin distribution seen in the mouse. Conservation of usherin is also seen at the nucleotide and amino acid level when comparing the mouse and human gene sequences. Evolutionary conservation of usherin expression at the molecular level and in tissues unaffected by Usher 2a supports the important structural and functional role this protein plays in the human. In addition, we believe that these results could lead to a diagnostic procedure for the detection of Usher syndrome and those who carry an USH2A mutation.

  10. Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

  11. Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade.

    Directory of Open Access Journals (Sweden)

    Elena Papaleo

    Full Text Available The ubiquitin (Ub system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3. E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7, which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.

  12. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    Science.gov (United States)

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

  13. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Directory of Open Access Journals (Sweden)

    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.

  14. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    Science.gov (United States)

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-08-10

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

  15. A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

    Science.gov (United States)

    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

    2011-02-01

    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

  16. The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

    Science.gov (United States)

    Gupta, Deepak K; Rühl, Martin; Mishra, Bagdevi; Kleofas, Vanessa; Hofrichter, Martin; Herzog, Robert; Pecyna, Marek J; Sharma, Rahul; Kellner, Harald; Hennicke, Florian; Thines, Marco

    2018-01-15

    Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

  17. Telomeric expression sites are highly conserved in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  18. High levels of maize in broiler diets with or without microbial enzyme ...

    African Journals Online (AJOL)

    mbhuiya3

    2013-03-13

    Mar 13, 2013 ... High levels of maize in broiler diets with or without microbial enzyme .... to improve carbohydrate digestion and availability of phosphorus from ... grinding machine and stored at –4 ºC in airtight containers for chemical analysis ...

  19. High-throughput screening for industrial enzyme production hosts by droplet microfluidics

    DEFF Research Database (Denmark)

    Sjostrom, Staffan L.; Bai, Yunpeng; Huang, Mingtao

    2014-01-01

    A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α......-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host...

  20. High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

    KAUST Repository

    Alam, Intikhab

    2016-01-26

    More and more genomes and metagenomes are being sequenced since the advent of Next Generation Sequencing Technologies (NGS). Many metagenomic samples are collected from a variety of environments, each exhibiting a different environmental profile, e.g. temperature, environmental chemistry, etc… These metagenomes can be profiled to unearth enzymes relevant to several industries based on specific enzyme properties such as ability to work on extreme conditions, such as extreme temperatures, salinity, anaerobically, etc.. In this work, we present the DMAP platform comprising of a high-throughput metagenomic annotation pipeline and a data-warehouse for comparisons and profiling across large number of metagenomes. We developed two reference databases for profiling of important genes, one containing enzymes related to different industries and the other containing genes with potential bioactivity roles. In this presentation we describe an example analysis of a large number of publicly available metagenomic sample from TARA oceans study (Science 2015) that covers significant part of world oceans.

  1. High-solids enrichment of thermophilic microbial communities and their enzymes on bioenergy feedstocks

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A. P.; Allgaier, M.; Singer, S.W.; Hazen, T.C.; Simmons, B.A.; Hugenholtz, P.; VanderGheynst, J.S.

    2011-04-01

    Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

  2. High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

    Science.gov (United States)

    Wu, Ching-Fang; Lee, Ching-Tai; Kuo, Yao-Hung; Chen, Tzu-Haw; Chang, Chi-Yang; Chang, I-Wei; Wang, Wen-Lun

    2017-09-01

    Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression

  3. Homologous high-throughput expression and purification of highly conserved E coli proteins

    Directory of Open Access Journals (Sweden)

    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  4. Visualization of conserved structures by fusing highly variable datasets.

    Science.gov (United States)

    Silverstein, Jonathan C; Chhadia, Ankur; Dech, Fred

    2002-01-01

    Reality (VR) environment. The accuracy of the fusions was determined qualitatively by comparing the transformed atlas overlaid on the appropriate CT. It was examined for where the transformed structure atlas was incorrectly overlaid (false positive) and where it was incorrectly not overlaid (false negative). According to this method, fusions 1 and 2 were correct roughly 50-75% of the time, while fusions 3 and 4 were correct roughly 75-100%. The CT dataset augmented with transformed dataset was viewed arbitrarily in user-centered perspective stereo taking advantage of features such as scaling, windowing and volumetric region of interest selection. This process of auto-coloring conserved structures in variable datasets is a step toward the goal of a broader, standardized automatic structure visualization method for radiological data. If successful it would permit identification, visualization or deletion of structures in radiological data by semi-automatically applying canonical structure information to the radiological data (not just processing and visualization of the data's intrinsic dynamic range). More sophisticated selection of control points and patterns of warping may allow for more accurate transforms, and thus advances in visualization, simulation, education, diagnostics, and treatment planning.

  5. Analytic, high β, flux conserving equilibria for cylindrical tokamaks

    International Nuclear Information System (INIS)

    Sigmar, D.J.; Vahala, G.

    1978-09-01

    Using Grad's theory of generalized differential equations, the temporal evolution from low to high β due to ''adiabatic'' and nonadiabatic (i.e., neutral beam injection) heating of a cylindrical tokamak plasma with circular cross section and peaked current profiles is calculated analytically. The influence of shaping the initial safety factor profile and the beam deposition profile and the effect of minor radius compression on the equilibrium is analyzed

  6. Analytic, high β, flux conserving equilibria for cylindrical tokamaks

    International Nuclear Information System (INIS)

    Sigmar, D.J.; Vahala, G.

    1978-01-01

    Using Grad's theory of generalized differential equations, the temporal evolution from low to high β due to ''adiabatic'' and nonadiabatic (i.e., neutral beam injection) heating of a cylindrical tokamak plasma with circular cross section and peaked current profiles is calculated analytically. The influence of shaping the initial safety factor profile and the beam deposition profile and the effect of minor radius compression on the equilibrium is analyzed

  7. Enzymic hydrolysis of xylans. I. A high xylanase and beta-xylosidase producing strain of Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Conrad, D.

    1981-01-01

    Aspergillus niger, strain 110.42 (CBS) was selected as a producer of high xylanolytic activities. The time course of xylanase and beta-xylosidase production as well as the effect of pH and temperature on the activity of these enzymes were studied. High-performance liquid chromatography analysis of the enzymic degradation of arabinoxylan showed a nearly complete conversion to pentose sugars. Aspects of using crude xylanase preparations for enzymic saccharification of xylans are discussed.

  8. Mining lipolytic enzymes in community DNA from high Andean soils using a targeted approach.

    Science.gov (United States)

    Borda-Molina, Daniel; Montaña, José Salvador; Zambrano, María Mercedes; Baena, Sandra

    2017-08-01

    Microbial enrichments cultures are a useful strategy to speed up the search for enzymes that can be employed in industrial processes. Lipases have gained special attention because they show unique properties such as: broad substrate specificity, enantio- and regio-selectivity and stability in organic solvents. A major goal is to identify novel lipolytic enzymes from microorganisms living in cold extreme environments such as high Andean soils, of relevance to our study being their capability be used in industrial processes. Paramo and glacier soils from the Nevados National Park in Colombia were sampled and microbial communities enriched through a fed-batch fermentation using olive oil as an inductor substrate. After 15 days of enrichment under aerobic conditions, total DNA was extracted. Subsequently, metagenomic libraries were constructed in the cosmid vector pWEB-TNC™. After functional screening, twenty and eighteen lipolytic clones were obtained from Paramo and Glacier soil enrichments, respectively. Based on lipid hydrolysis halo dimensions, the clone (Gla1) from a glacier enrichment was selected. A gene related to lipolytic activity was subcloned to evaluate enzyme properties. Phylogenetic analysis of the identified gene showed that the encoded lipase belongs to the family GDSL from a Ralstonia-like species. Interestingly, the secreted enzyme exhibited stability at high temperature and alkaline conditions, specifically the preferred conditions at 80 °C and pH 9.0. Thus, with the identification of an enzyme with non-expected properties, in this study is shown the potential of extreme cold environments to be explored for new catalytic molecules, using current molecular biology techniques, with applications in industrial processes, which demand stability under harsh conditions.

  9. A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

    Science.gov (United States)

    Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

    2017-12-08

    Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

    Energy Technology Data Exchange (ETDEWEB)

    Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

    2013-03-22

    Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

  11. High-resolution satellite imagery is an important yet underutilized resource in conservation biology.

    Science.gov (United States)

    Boyle, Sarah A; Kennedy, Christina M; Torres, Julio; Colman, Karen; Pérez-Estigarribia, Pastor E; de la Sancha, Noé U

    2014-01-01

    Technological advances and increasing availability of high-resolution satellite imagery offer the potential for more accurate land cover classifications and pattern analyses, which could greatly improve the detection and quantification of land cover change for conservation. Such remotely-sensed products, however, are often expensive and difficult to acquire, which prohibits or reduces their use. We tested whether imagery of high spatial resolution (≤5 m) differs from lower-resolution imagery (≥30 m) in performance and extent of use for conservation applications. To assess performance, we classified land cover in a heterogeneous region of Interior Atlantic Forest in Paraguay, which has undergone recent and dramatic human-induced habitat loss and fragmentation. We used 4 m multispectral IKONOS and 30 m multispectral Landsat imagery and determined the extent to which resolution influenced the delineation of land cover classes and patch-level metrics. Higher-resolution imagery more accurately delineated cover classes, identified smaller patches, retained patch shape, and detected narrower, linear patches. To assess extent of use, we surveyed three conservation journals (Biological Conservation, Biotropica, Conservation Biology) and found limited application of high-resolution imagery in research, with only 26.8% of land cover studies analyzing satellite imagery, and of these studies only 10.4% used imagery ≤5 m resolution. Our results suggest that high-resolution imagery is warranted yet under-utilized in conservation research, but is needed to adequately monitor and evaluate forest loss and conversion, and to delineate potentially important stepping-stone fragments that may serve as corridors in a human-modified landscape. Greater access to low-cost, multiband, high-resolution satellite imagery would therefore greatly facilitate conservation management and decision-making.

  12. The effect of angiotensin converting enzyme genotype on aerobic capacity following high intensity interval training

    OpenAIRE

    Goddard, N; Baker, M.D; Higgins, T; Cobbold, C

    2014-01-01

    Obesity increases the risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Physical activity can reduce T2DM and CVD risk, and increase aerobic capacity, a significant predictor of all-cause mortality and morbidity. High intensity interval training (HIIT) produces similar improvements in aerobic capacity to continuous moderate exercise (CME). Different genotypes of angiotensin converting enzyme (ACE) have been implicated in improving aerobic capacity and theref...

  13. Breast-conservation treatment without any surgical procedure using new enzyme-targeting radiosensitization treatment for aged and/or op. refused patients with breast cancer

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Kubota, Kei; Miyatake, Kana

    2008-01-01

    We developed a new radiosensitizer containing hydrogen peroxide and sodium hyaluronate for topical tumor injection for various types of tumors, and the method was named KORTUC II (Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas, Type II). KORTUC II trial was accepted by our local ethical committee concerning of the injection for advanced skin cancer, advanced bone/soft tissue malignant neoplasms, breast cancer of op refused or aged patients, and metastatic lymph nodes. Concerning breast cancer, ten patients were enrolled in the KORTUC II trial upon fully informed consent. All of them showed clinically complete response by the new enzyme-targeting radiosensitization treatment (KORTUC II) without any severe complications excluding mild dermatitis (grade I). Nine of the 10 patients have so far shown neither local recurrence nor distant metastasis, and the mean follow-up period at the end of December 2007 was still short and approximately 12 months. Especially for patients with breast cancer, breast-conservation treatment without any surgical procedure can be performed by using our new radiosensitizer for topical injection into the tumor tissue. (author)

  14. A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

    Science.gov (United States)

    Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

    2016-11-01

    The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

  15. High throughput, high resolution enzymatic lithography process: effect of crystallite size, moisture, and enzyme concentration.

    Science.gov (United States)

    Mao, Zhantong; Ganesh, Manoj; Bucaro, Michael; Smolianski, Igor; Gross, Richard A; Lyons, Alan M

    2014-12-08

    By bringing enzymes into contact with predefined regions of a surface, a polymer film can be selectively degraded to form desired patterns that find a variety of applications in biotechnology and electronics. This so-called "enzymatic lithography" is an environmentally friendly process as it does not require actinic radiation or synthetic chemicals to develop the patterns. A significant challenge to using enzymatic lithography has been the need to restrict the mobility of the enzyme in order to maintain control of feature sizes. Previous approaches have resulted in low throughput and were limited to polymer films only a few nanometers thick. In this paper, we demonstrate an enzymatic lithography system based on Candida antartica lipase B (CALB) and poly(ε-caprolactone) (PCL) that can resolve fine-scale features, (<1 μm across) in thick (0.1-2.0 μm) polymer films. A Polymer Pen Lithography (PPL) tool was developed to deposit an aqueous solution of CALB onto a spin-cast PCL film. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 μm). The effect of printing conditions and relative humidity during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a "Green" approach to the fabrication of printed circuit boards.

  16. High similarity of phylogenetic profiles of rate-limiting enzymes with inhibitory relation in Human, Mouse, Rat, budding Yeast and E. coli.

    Science.gov (United States)

    Zhao, Min; Qu, Hong

    2011-11-30

    The phylogenetic profile is widely used to characterize functional linkage and conservation between proteins without amino acid sequence similarity. To survey the conservative regulatory properties of rate-limiting enzymes (RLEs) in metabolic inhibitory network across different species, we define the enzyme inhibiting pair as: where the first enzyme in a pair is the inhibitor provider and the second is the target of the inhibitor. Phylogenetic profiles of enzymes in the inhibiting pairs are further generated to measure the functional linkage of these enzymes during evolutionary history. We find that the RLEs generate, on average, over half of all in vivo inhibitors in each surveyed model organism. And these inhibitors inhibit on average over 85% targets in metabolic inhibitory network and cover the majority of targets of cross-pathway inhibiting relations. Furthermore, we demonstrate that the phylogenetic profiles of the enzymes in inhibiting pairs in which at least one enzyme is rate-limiting often show higher similarities than those in common inhibiting enzyme pairs. In addition, RLEs, compared to common metabolic enzymes, often tend to produce ADP instead of AMP in conservative inhibitory networks. Combined with the conservative roles of RLEs in their efficiency in sensing metabolic signals and transmitting regulatory signals to the rest of the metabolic system, the RLEs may be important molecules in balancing energy homeostasis via maintaining the ratio of ATP to ADP in living cells. Furthermore, our results indicate that similarities of phylogenetic profiles of enzymes in the inhibiting enzyme pairs are not only correlated with enzyme topological importance, but also related with roles of the enzymes in metabolic inhibitory network.

  17. High-throughput sequencing, characterization and detection of new and conserved cucumber miRNAs.

    Directory of Open Access Journals (Sweden)

    Germán Martínez

    Full Text Available Micro RNAS (miRNAs are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome.In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

  18. High-Order Entropy Stable Finite Difference Schemes for Nonlinear Conservation Laws: Finite Domains

    Science.gov (United States)

    Fisher, Travis C.; Carpenter, Mark H.

    2013-01-01

    Developing stable and robust high-order finite difference schemes requires mathematical formalism and appropriate methods of analysis. In this work, nonlinear entropy stability is used to derive provably stable high-order finite difference methods with formal boundary closures for conservation laws. Particular emphasis is placed on the entropy stability of the compressible Navier-Stokes equations. A newly derived entropy stable weighted essentially non-oscillatory finite difference method is used to simulate problems with shocks and a conservative, entropy stable, narrow-stencil finite difference approach is used to approximate viscous terms.

  19. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks

    Directory of Open Access Journals (Sweden)

    Casteleijn Marco G

    2008-11-01

    Full Text Available Abstract Background Here we describe a novel cultivation method, called EnBase™, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries. Results High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel. Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3 resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method. Conclusion The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The

  20. Spatial overlap between environmental policy instruments and areas of high conservation value in forest.

    Science.gov (United States)

    Sverdrup-Thygeson, Anne; Søgaard, Gunnhild; Rusch, Graciela M; Barton, David N

    2014-01-01

    In order to safeguard biodiversity in forest we need to know how forest policy instruments work. Here we use a nationwide network of 9400 plots in productive forest to analyze to what extent large-scale policy instruments, individually and together, target forest of high conservation value in Norway. We studied both instruments working through direct regulation; Strict Protection and Landscape Protection, and instruments working through management planning and voluntary schemes of forest certification; Wilderness Area and Mountain Forest. As forest of high conservation value (HCV-forest) we considered the extent of 12 Biodiversity Habitats and the extent of Old-Age Forest. We found that 22% of productive forest area contained Biodiversity Habitats. More than 70% of this area was not covered by any large-scale instruments. Mountain Forest covered 23%, while Strict Protection and Wilderness both covered 5% of the Biodiversity Habitat area. A total of 9% of productive forest area contained Old-Age Forest, and the relative coverage of the four instruments was similar as for Biodiversity Habitats. For all instruments, except Landscape Protection, the targeted areas contained significantly higher proportions of HCV-forest than areas not targeted by these instruments. Areas targeted by Strict Protection had higher proportions of HCV-forest than areas targeted by other instruments, except for areas targeted by Wilderness Area which showed similar proportions of Biodiversity Habitats. There was a substantial amount of spatial overlap between the policy tools, but no incremental conservation effect of overlapping instruments in terms of contributing to higher percentages of targeted HCV-forest. Our results reveal that although the current policy mix has an above average representation of forest of high conservation value, the targeting efficiency in terms of area overlap is limited. There is a need to improve forest conservation and a potential to cover this need by better

  1. Prospects for Parity Non-conservation Experiments with Highly Charged Heavy Ions

    OpenAIRE

    Maul, M.; Schäfer, A.; Greiner, W.; Indelicato, P.

    1996-01-01

    We discuss the prospects for parity non-conservation experiments with highly charged heavy ions. Energy levels and parity mixing for heavy ions with two to five electrons are calculated. We investigate two-photon-transitions and the possibility to observe interference effects between weak-matrix elements and Stark matrix elements for periodic electric field configurations.

  2. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

    Science.gov (United States)

    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

  3. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

    Directory of Open Access Journals (Sweden)

    Mareš Michael

    2008-03-01

    Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

  4. Balancing forest-regeneration probabilities and maintenance costs in dry grasslands of high conservation priority

    Science.gov (United States)

    Bolliger, Janine; Edwards, Thomas C.; Eggenberg, Stefan; Ismail, Sascha; Seidl, Irmi; Kienast, Felix

    2011-01-01

    Abandonment of agricultural land has resulted in forest regeneration in species-rich dry grasslands across European mountain regions and threatens conservation efforts in this vegetation type. To support national conservation strategies, we used a site-selection algorithm (MARXAN) to find optimum sets of floristic regions (reporting units) that contain grasslands of high conservation priority. We sought optimum sets that would accommodate 136 important dry-grassland species and that would minimize forest regeneration and costs of management needed to forestall predicted forest regeneration. We did not consider other conservation elements of dry grasslands, such as animal species richness, cultural heritage, and changes due to climate change. Optimal sets that included 95–100% of the dry grassland species encompassed an average of 56–59 floristic regions (standard deviation, SD 5). This is about 15% of approximately 400 floristic regions that contain dry-grassland sites and translates to 4800–5300 ha of dry grassland out of a total of approximately 23,000 ha for the entire study area. Projected costs to manage the grasslands in these optimum sets ranged from CHF (Swiss francs) 5.2 to 6.0 million/year. This is only 15–20% of the current total estimated cost of approximately CHF30–45 million/year required if all dry grasslands were to be protected. The grasslands of the optimal sets may be viewed as core sites in a national conservation strategy.

  5. Technical evaluation on high aging, and performance conditions on long-term conservation program

    International Nuclear Information System (INIS)

    Yamashita, Atsushi

    2001-01-01

    In order to secure safety and safe operation of power plants, in every nuclear power plants, conservation actions based on preventive conservation are performed. They contain operative condition monitoring, patrolling inspection, and periodical tests on important systems and apparatus by operators under plant operation and condition monitoring by maintenance workers, and so on, and when finding out their abnormal conditions, their detailed survey is performed to adopt adequate countermeasures such as recovery, exchange, and so on. And, to equipments for nuclear power generation periodical conditions were obliged by legal examinations and by independent inspections. As a result of these conservation actions, even on a plant elapsed about 30 years since beginning of its operation it was thought that the plant was aged with elapsing time even if not recognizing any indication on its aged deterioration at that time. Therefore, for its concrete countermeasure, by supposing long-term operation of a plant with longer operation history, some technical evaluation on aged phenomena were carried out, to investigate on reflection of the obtained results to present conservation actions. Here were described on efforts on the high aging countermeasures, and performing conditions of long-term conservation in the Tsuruga Unit No. 1 Nuclear Power Station. (G.K.)

  6. High qualitative and quantitative conservation of alternative splicing in Caenorhabditis elegans and Caenorhabditis briggsae

    DEFF Research Database (Denmark)

    Rukov, Jakob Lewin; Irimia, Manuel; Mørk, Søren

    2007-01-01

    Alternative splicing (AS) is an important contributor to proteome diversity and is regarded as an explanatory factor for the relatively low number of human genes compared with less complex animals. To assess the evolutionary conservation of AS and its developmental regulation, we have investigated...... the qualitative and quantitative expression of 21 orthologous alternative splice events through the development of 2 nematode species separated by 85-110 Myr of evolutionary time. We demonstrate that most of these alternative splice events present in Caenorhabditis elegans are conserved in Caenorhabditis briggsae....... Moreover, we find that relative isoform expression levels vary significantly during development for 78% of the AS events and that this quantitative variation is highly conserved between the 2 species. Our results suggest that AS is generally tightly regulated through development and that the regulatory...

  7. A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors

    Science.gov (United States)

    Guo, Jingxing; Zhang, Tao; Hu, Chengguo; Fu, Lei

    2015-01-01

    In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme

  8. High sensitivity, high surface area Enzyme-linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Singh, Harpal; Morita, Takahiro; Suzuki, Yuma; Shimojima, Masayuki; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

  9. Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

    Science.gov (United States)

    Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

    2012-01-01

    Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

  10. High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

    KAUST Repository

    Alam, Intikhab

    2016-01-01

    .g. temperature, environmental chemistry, etc… These metagenomes can be profiled to unearth enzymes relevant to several industries based on specific enzyme properties such as ability to work on extreme conditions, such as extreme temperatures, salinity

  11. Genes involved in complex adaptive processes tend to have highly conserved upstream regions in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Kohane Isaac

    2005-11-01

    Full Text Available Abstract Background Recent advances in genome sequencing suggest a remarkable conservation in gene content of mammalian organisms. The similarity in gene repertoire present in different organisms has increased interest in studying regulatory mechanisms of gene expression aimed at elucidating the differences in phenotypes. In particular, a proximal promoter region contains a large number of regulatory elements that control the expression of its downstream gene. Although many studies have focused on identification of these elements, a broader picture on the complexity of transcriptional regulation of different biological processes has not been addressed in mammals. The regulatory complexity may strongly correlate with gene function, as different evolutionary forces must act on the regulatory systems under different biological conditions. We investigate this hypothesis by comparing the conservation of promoters upstream of genes classified in different functional categories. Results By conducting a rank correlation analysis between functional annotation and upstream sequence alignment scores obtained by human-mouse and human-dog comparison, we found a significantly greater conservation of the upstream sequence of genes involved in development, cell communication, neural functions and signaling processes than those involved in more basic processes shared with unicellular organisms such as metabolism and ribosomal function. This observation persists after controlling for G+C content. Considering conservation as a functional signature, we hypothesize a higher density of cis-regulatory elements upstream of genes participating in complex and adaptive processes. Conclusion We identified a class of functions that are associated with either high or low promoter conservation in mammals. We detected a significant tendency that points to complex and adaptive processes were associated with higher promoter conservation, despite the fact that they have emerged

  12. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2014-01-01

    Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  13. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    Science.gov (United States)

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  14. Angiotensin-converting enzyme (ACE) alleles in the Quechua, a high altitude South American native population.

    Science.gov (United States)

    Rupert, J L; Devine, D V; Monsalve, M V; Hochachka, P W

    1999-01-01

    Recently it was reported that an allelic variant of the gene encoding angiotensin-converting enzyme (ACE) was significantly over-represented in a cohort of elite British mountaineers. It was proposed that this may be evidence for a specific genetic factor influencing the human capacity for physical performance. The implication that this allele could enhance performance at high altitude prompted us to determine its frequency in Quechua speaking natives living at altitudes greater than 3000m on the Andean Altiplano in South America. We found that the frequency of the putative performance allele in the Quechuas, although significantly higher than in Caucasians, was not different from lowland Native American populations. This observation suggests that, although the higher frequency of the 'performance allele' may have facilitated the migration of the ancestral Quechua to the highlands, the ACE insertion allele has not been subsequently selected for in this high altitude population.

  15. A highly sensitive electrochemical glucose sensor structuring with nickel hydroxide and enzyme glucose oxidase

    International Nuclear Information System (INIS)

    Mathew, Manjusha; Sandhyarani, N.

    2013-01-01

    Graphical abstract: A combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has successfully been exploited for the realization of a highly sensitive glucose sensor for the first time. -- Highlights: • A multilayered glucose biosensor with enhanced sensitivity was fabricated. • Combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has been exploited for the first time. • Exhibits a lower detection limit of 100 nM with a high sensitivity of 16,840 μA mM −1 cm −2 . • The surface shows a low Michaelis–Menten constant value of 2.4 μM. • Detailed mechanism of sensing was proposed and justified. -- Abstract: A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH) 2 ) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH) 2 deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 μM to 100 μM with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 μM indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications

  16. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  17. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II

    Directory of Open Access Journals (Sweden)

    Kanchanmala Deshpande

    2010-06-01

    Full Text Available A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx and horseradish peroxidase (HRP for determination of mercury Hg(II in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II was found to be linear in the range 5–500 pg.mL−1 with 3% CVin inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II was measurable as low as 1 pg.mL−1 within15 min. About 10-fold more specificity of the developed assay for Hg(II analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II and lead Pb(II. Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II, Cd(II and Pb(II, inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%.

  18. Fabrication of CuO nanoplatelets for highly sensitive enzyme-free determination of glucose

    Energy Technology Data Exchange (ETDEWEB)

    Wang Juan [School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640 (China); Zhang Weide, E-mail: zhangwd@scut.edu.cn [School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640 (China)

    2011-09-01

    Highlights: > Adhered growth of CuO nanoplatelets on Cu foils. > Enzyme-free glucose sensor with very high sensitivity. > Excellent stability and good anti-interference ability. - Abstract: CuO nanoplatelets were grown on Cu foils by a one step, template free process. The structure and morphology of the CuO nanoplatelets were characterized by X-ray diffraction, scanning and transmission electron microscopy. The CuO nanoplatelets grown on Cu foil were integrated to be an electrode for glucose sensing. The electrocatalytic activity of the CuO nanoplatelets electrode for glucose in alkaline media was investigated by cyclic voltammetry and chronoamperometry. The electrode exhibits a sensitivity of 3490.7 {mu}A mM{sup -1} cm{sup -2} to glucose which is much higher than that of most reported enzyme-free glucose sensors and the linear range was obtained over a concentration up to 0.80 mM with a detection limit of 0.50 {mu}M (signal/noise = 3). Exhilaratingly, the electrode based on the CuO nanoplatelets is resistant against poisoning by chloride ion, and the interference from the oxidation of common interfering species, such as uric acid, ascorbic acid, dopamine and carbonhydrate compounds, can also be effectively avoided. Finally, the electrode was applied to analyze glucose concentration in human serum samples.

  19. Conservative Management for Stable High Ankle Injuries in Professional Football Players.

    Science.gov (United States)

    Knapik, Derrick M; Trem, Anthony; Sheehan, Joseph; Salata, Michael J; Voos, James E

    High ankle "syndesmosis" injuries are common in American football players relative to the general population. At the professional level, syndesmotic sprains represent a challenging and unique injury lacking a standardized rehabilitation protocol during conservative management. PubMed, Biosis Preview, SPORTDiscus, PEDro, and EMBASE databases were searched using the terms syndesmotic injuries, American football, conservative management, and rehabilitation. Clinical review. Level 3. When compared with lateral ankle sprains, syndesmosis injuries result in significantly prolonged recovery times and games lost. For stable syndesmotic injuries, conservative management features a brief period of immobilization and protected weightbearing followed by progressive strengthening exercises and running, and athletes can expect to return to competition in 2 to 6 weeks. Further research investigating the efficacy of dry needling and blood flow restriction therapy is necessary to evaluate the benefit of these techniques in the rehabilitation process. Successful conservative management of stable syndesmotic injuries in professional American football athletes requires a thorough understanding of the anatomy, injury mechanisms, diagnosis, and rehabilitation strategies utilized in elite athletes.

  20. Enhancing Conservation with High Resolution Productivity Datasets for the Conterminous United States

    Science.gov (United States)

    Robinson, Nathaniel Paul

    Human driven alteration of the earth's terrestrial surface is accelerating through land use changes, intensification of human activity, climate change, and other anthropogenic pressures. These changes occur at broad spatio-temporal scales, challenging our ability to effectively monitor and assess the impacts and subsequent conservation strategies. While satellite remote sensing (SRS) products enable monitoring of the earth's terrestrial surface continuously across space and time, the practical applications for conservation and management of these products are limited. Often the processes driving ecological change occur at fine spatial resolutions and are undetectable given the resolution of available datasets. Additionally, the links between SRS data and ecologically meaningful metrics are weak. Recent advances in cloud computing technology along with the growing record of high resolution SRS data enable the development of SRS products that quantify ecologically meaningful variables at relevant scales applicable for conservation and management. The focus of my dissertation is to improve the applicability of terrestrial gross and net primary productivity (GPP/NPP) datasets for the conterminous United States (CONUS). In chapter one, I develop a framework for creating high resolution datasets of vegetation dynamics. I use the entire archive of Landsat 5, 7, and 8 surface reflectance data and a novel gap filling approach to create spatially continuous 30 m, 16-day composites of the normalized difference vegetation index (NDVI) from 1986 to 2016. In chapter two, I integrate this with other high resolution datasets and the MOD17 algorithm to create the first high resolution GPP and NPP datasets for CONUS. I demonstrate the applicability of these products for conservation and management, showing the improvements beyond currently available products. In chapter three, I utilize this dataset to evaluate the relationships between land ownership and terrestrial production

  1. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

    Science.gov (United States)

    Hamilton, Natasha A; Tammen, Imke; Raadsma, Herman W

    2013-01-01

    Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  2. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

    Directory of Open Access Journals (Sweden)

    Natasha A Hamilton

    Full Text Available Angiotensin converting enzyme (ACE is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  3. Response of broiler chickens to diets containing artificially dried high-moisture maize supplemented with microbial enzymes

    OpenAIRE

    Bhuiyan, M.M; Islam, A.F; Iji, P.A

    2010-01-01

    The effect of feeding high-moisture maize grains dried in the sun or artificially in a forced draught oven at 80, 90 or 100 ºC for 24 hours and supplemented with microbial enzymes (Avizyme 1502 and Phyzyme XP) on growth performance, visceral organs, tissue protein, enzyme activity and gut development was investigated in a broiler growth trial. Feed intake (FI) up to 21 days decreased as a results of oven drying of grains whereas supplementation with microbial enzymes increased FI compared to ...

  4. Entropy Viscosity Method for High-Order Approximations of Conservation Laws

    KAUST Repository

    Guermond, J. L.

    2010-09-17

    A stabilization technique for conservation laws is presented. It introduces in the governing equations a nonlinear dissipation function of the residual of the associated entropy equation and bounded from above by a first order viscous term. Different two-dimensional test cases are simulated - a 2D Burgers problem, the "KPP rotating wave" and the Euler system - using high order methods: spectral elements or Fourier expansions. Details on the tuning of the parameters controlling the entropy viscosity are given. © 2011 Springer.

  5. Entropy Viscosity Method for High-Order Approximations of Conservation Laws

    KAUST Repository

    Guermond, J. L.; Pasquetti, R.

    2010-01-01

    A stabilization technique for conservation laws is presented. It introduces in the governing equations a nonlinear dissipation function of the residual of the associated entropy equation and bounded from above by a first order viscous term. Different two-dimensional test cases are simulated - a 2D Burgers problem, the "KPP rotating wave" and the Euler system - using high order methods: spectral elements or Fourier expansions. Details on the tuning of the parameters controlling the entropy viscosity are given. © 2011 Springer.

  6. Bacterial Glycosyltransferases: Challenges and opportunities of a highly diverse enzyme class toward tailoring natural products

    Directory of Open Access Journals (Sweden)

    Jochen eSchmid

    2016-02-01

    Full Text Available The enzyme subclass of glycosyltransferases (EC 2.4 currently comprises 97 families as specified by CAZy classification. One of their important roles is in the biosynthesis of disaccharides, oligosaccharides and polysaccharides by catalyzing the transfer of sugar moieties from activated donor molecules to other sugar molecules. In addition glycosyltransferases also catalyze the transfer of sugar moieties onto aglycons, which is of great relevance for the synthesis of many high value natural products. Bacterial glycosyltransferases show a higher sequence similarity in comparison to mammalian ones. Even when most glycosyltransferases are poorly explored, state of the art technologies, such as protein engineering, domain swapping or computational analysis strongly enhance our understanding and utilization of these very promising classes of proteins. This perspective article will focus on bacterial glycosyltransferases, especially on classification, screening and engineering strategies to alter substrate specificity. The future development in these fields as well as obstacles and challenges will be highlighted and discussed.

  7. Highly conserved non-coding sequences are associated with vertebrate development.

    Directory of Open Access Journals (Sweden)

    Adam Woolfe

    2005-01-01

    Full Text Available In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH, in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development

  8. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development

    Science.gov (United States)

    Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823

  9. Fungal Enzymes and Yeasts for Conversion of Plant Biomass to Bioenergy and High-Value Products

    DEFF Research Database (Denmark)

    Lange, Lene

    2017-01-01

    Fungi and fungal enzymes play important roles in the new bioeconomy. Enzymes from filamentous fungi can unlock the potential of recalcitrant lignocellulose structures of plant cell walls as a new resource, and fungi such as yeast can produce bioethanol from the sugars released after enzyme treatm...... contributed to mycology and environmental research? Future perspectives and approaches are listed, highlighting the importance of fungi in development of the bioeconomy.......Fungi and fungal enzymes play important roles in the new bioeconomy. Enzymes from filamentous fungi can unlock the potential of recalcitrant lignocellulose structures of plant cell walls as a new resource, and fungi such as yeast can produce bioethanol from the sugars released after enzyme...... treatment. Such processes reflect inherent characteristics of the fungal way of life, namely, that fungi as heterotrophic organisms must break down complex carbon structures of organic materials to satisfy their need for carbon and nitrogen for growth and reproduction. This chapter describes major steps...

  10. Study of High Sensitive-CRP and Cardiac Marker Enzymes in Acute Coronary Syndrome

    Directory of Open Access Journals (Sweden)

    Srikrishna R,

    2015-04-01

    Full Text Available Background: Inflammation has been proposed as a contributor to different stages in the pathogenesis of Coronary Heart Disease (CHD. High sensitive C-Reactive Protein (hs-CRP, an acute-phase plasma protein synthesized by the liver, is the most extensively studied systemic marker of inflammation. Elevated hsCRP concentrations early in Acute Coronary Syndrome (ACS, prior to the tissue necrosis, may be a surrogate marker for cardiovascular co-morbidities. The cardiac marker enzymes Creatine Kinase myocardial bound (CK-MB, Aspartate Aminotransferase (AST and lactate dehydrogenase (LDH have been known to be increased in coronary artery diseases. Objective: The aim of the study was to measure hs-CRP levels and other cardiac marker enzymes in ACS patients and to compare the levels of hs-CRP with other cardiac marker enzymes between ST Elevation Myocardial Infarction (STEMI and Non-ST Elevation Myocardial Infarction (NSTEMI patients. Material and Methods: The study group consisted of 207 consecutive patients admitted to Sri Siddhartha Medical College Hospital within the first 6 hours from the onset of chest pain. Patients were diagnosed as Unstable Angina (UA, (n=84; STEMI (n=63 and NSTEMI (n=60. ACS patients were compared with 211 healthy age and sex matched controls. Hs-CRP, CK-MB, AST and LDH levels were measured by standard methods in both groups at baseline and forcases at 36-48 hours i.e. Peak levels. Results: ACS patients had significantly (p<0.05 higher levels of hs-CRP, CKMB, AST and LDH in comparison to controls at baseline. Hs-CRP, CK-MB, AST and LDH levels were significantly higher in STEMI patients compared to NSTEMI patients (p<0.05 at baseline. There was a significant difference regarding peak hs-CRP levels between the two groups, as STEMI patients had significantly higher peak hs-CRP levels compared to NSTEMI patients (p<0.05. Conclusion: STEMI patients have significantly higher peak hsCRP levels compared to NSTEMI patients. These data

  11. A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

    Science.gov (United States)

    Lam, Sonia Y; Yeung, Rachel C Y; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

    2011-03-01

    Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

  12. A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

    Directory of Open Access Journals (Sweden)

    Sonia Y Lam

    2011-03-01

    Full Text Available Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy.Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

  13. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    Science.gov (United States)

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains

    Directory of Open Access Journals (Sweden)

    Wang Yiguo

    2008-10-01

    Full Text Available Abstract Background Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs. Accurate prediction of SLiMs has been difficult because they are short (often Results Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. Conclusion The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains.

  15. A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

    Science.gov (United States)

    Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

    1999-01-01

    It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

  16. High levels of maize in broiler diets with or without microbial enzyme ...

    African Journals Online (AJOL)

    Over the feeding period (21 d), there was an increase in feed intake as maize inclusion level (MIL) increased in diets, while supplementation with microbial enzyme improved feed intake only in the MM diet. There was an improvement in live weight (LW) in chickens with increased MIL in their diets. The microbial enzyme ...

  17. In-situ nanoelectrospray for high-throughput screening of enzymes and real-time monitoring of reactions.

    Science.gov (United States)

    Yang, Yuhan; Han, Feifei; Ouyang, Jin; Zhao, Yunling; Han, Juan; Na, Na

    2016-01-01

    The in-situ and high-throughput evaluation of enzymes and real-time monitoring of enzyme catalyzed reactions in liquid phase is quite significant in the catalysis industry. In-situ nanoelectrospray, the direct sampling and ionization method for mass spectrometry, has been applied for high-throughput evaluation of enzymes, as well as the on-line monitoring of reactions. Simply inserting a capillary into a liquid system with high-voltage applied, analytes in liquid reaction system can be directly ionized at the capillary tip with small volume consumption. With no sample pre-treatment or injection procedure, different analytes such as saccharides, amino acids, alkaloids, peptides and proteins can be rapidly and directly extracted from liquid phase and ionized at the capillary tip. Taking irreversible transesterification reaction of vinyl acetate and ethanol as an example, this technique has been used for the high-throughput evaluation of enzymes, fast optimizations, as well as real-time monitoring of reaction catalyzed by different enzymes. In addition, it is even softer than traditional electrospray ionization. The present method can also be used for the monitoring of other homogenous and heterogeneous reactions in liquid phases, which will show potentials in the catalysis industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Species Richness and Community Structure on a High Latitude Reef: Implications for Conservation and Management

    Directory of Open Access Journals (Sweden)

    Wayne Houston

    2011-07-01

    Full Text Available In spite of the wealth of research on the Great Barrier Reef, few detailed biodiversity assessments of its inshore coral communities have been conducted. Effective conservation and management of marine ecosystems begins with fine-scale biophysical assessments focused on diversity and the architectural species that build the structural framework of the reef. In this study, we investigate key coral diversity and environmental attributes of an inshore reef system surrounding the Keppel Bay Islands near Rockhampton in Central Queensland, Australia, and assess their implications for conservation and management. The Keppels has much higher coral diversity than previously found. The average species richness for the 19 study sites was ~40 with representatives from 68% of the ~244 species previously described for the southern Great Barrier Reef. Using scleractinian coral species richness, taxonomic distinctiveness and coral cover as the main criteria, we found that five out of 19 sites had particularly high conservation value. A further site was also considered to be of relatively high value. Corals at this site were taxonomically distinct from the others (representatives of two families were found here but not at other sites and a wide range of functionally diverse taxa were present. This site was associated with more stressful conditions such as high temperatures and turbidity. Highly diverse coral communities or biodiversity ‘hotspots’ and taxonomically distinct reefs may act as insurance policies for climatic disturbance, much like Noah’s Arks for reefs. While improving water quality and limiting anthropogenic impacts are clearly important management initiatives to improve the long-term outlook for inshore reefs, identifying, mapping and protecting these coastal ‘refugia’ may be the key for ensuring their regeneration against catastrophic climatic disturbance in the meantime.

  19. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    Science.gov (United States)

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  20. Impact of local empowerment on conservation practices in a highly developed country

    OpenAIRE

    Engen, Sigrid; Hausner, Vera Helene

    2017-01-01

    Source at http://dx.doi.org/10.1111/conl.12369 Community-based conservation, where local decision makers are responsible for balancing conservation and development, is often preferred to exclusion- ary conservation that prioritizes use-limitation through strict regulation. Un- raveling the evidence for conservation impact of different governance regimes is challenging. Focusing on conservation practices before and after a reform can provide an early indication of behaviora...

  1. Use of ancient sedimentary DNA as a novel conservation tool for high-altitude tropical biodiversity.

    Science.gov (United States)

    Boessenkool, Sanne; McGlynn, Gayle; Epp, Laura S; Taylor, David; Pimentel, Manuel; Gizaw, Abel; Nemomissa, Sileshi; Brochmann, Christian; Popp, Magnus

    2014-04-01

    Conservation of biodiversity may in the future increasingly depend upon the availability of scientific information to set suitable restoration targets. In traditional paleoecology, sediment-based pollen provides a means to define preanthropogenic impact conditions, but problems in establishing the exact provenance and ecologically meaningful levels of taxonomic resolution of the evidence are limiting. We explored the extent to which the use of sedimentary ancient DNA (sedaDNA) may complement pollen data in reconstructing past alpine environments in the tropics. We constructed a record of afro-alpine plants retrieved from DNA preserved in sediment cores from 2 volcanic crater sites in the Albertine Rift, eastern Africa. The record extended well beyond the onset of substantial anthropogenic effects on tropical mountains. To ensure high-quality taxonomic inference from the sedaDNA sequences, we built an extensive DNA reference library covering the majority of the afro-alpine flora, by sequencing DNA from taxonomically verified specimens. Comparisons with pollen records from the same sediment cores showed that plant diversity recovered with sedaDNA improved vegetation reconstructions based on pollen records by revealing both additional taxa and providing increased taxonomic resolution. Furthermore, combining the 2 measures assisted in distinguishing vegetation change at different geographic scales; sedaDNA almost exclusively reflects local vegetation, whereas pollen can potentially originate from a wide area that in highlands in particular can span several ecozones. Our results suggest that sedaDNA may provide information on restoration targets and the nature and magnitude of human-induced environmental changes, including in high conservation priority, biodiversity hotspots, where understanding of preanthropogenic impact (or reference) conditions is highly limited. © 2013 Society for Conservation Biology.

  2. Effect of high dietary copper on growth, antioxidant and lipid metabolism enzymes of juvenile larger yellow croaker Larimichthys croceus

    Directory of Open Access Journals (Sweden)

    Fanxing Meng

    2016-05-01

    Full Text Available A study was carried out to test the responses of juvenile larger yellow croaker Larimichthys croceus to high Cu intake. Experimental diets were formulated containing three levels of Cu: low Cu (3.67 mg/kg, middle Cu (13.65 mg/kg and high Cu (25.78 mg/kg, and each diet were fed to large yellow croaker in triplicate for 10 weeks. Final body weight, weight gain and feed intake were the lowest in high Cu group, but hepatosomatic index was the highest; Cu concentrations in the whole-body, muscle and liver of fish fed low Cu diet was the lowest; Liver superoxide dismutase, catalase and glutathione peroxidase activities in fish fed high Cu diet were lower than those in fish fed other diets; The higher content of liver thiobarbituric acid reactive substance content was found in high Cu group, followed by middle Cu group, and the lowest in low Cu group; Liver 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase and fatty acid synthase activities were the lowest in high Cu group, but lipoprotein lipase activity was the highest. This study indicated that high copper intake reduced growth of juvenile larger yellow croaker, inhibited activities of antioxidant enzymes and lipid synthetases, and led to energy mobilization. Keywords: Larger yellow croaker, Copper, Antioxidant enzyme, Lipid metabolism enzyme

  3. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

    2011-01-01

    Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines....... With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites...... between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines...

  4. Low-Intensity Agricultural Landscapes in Transylvania Support High Butterfly Diversity: Implications for Conservation

    Science.gov (United States)

    Loos, Jacqueline; Dorresteijn, Ine; Hanspach, Jan; Fust, Pascal; Rakosy, László; Fischer, Joern

    2014-01-01

    European farmland biodiversity is declining due to land use changes towards agricultural intensification or abandonment. Some Eastern European farming systems have sustained traditional forms of use, resulting in high levels of biodiversity. However, global markets and international policies now imply rapid and major changes to these systems. To effectively protect farmland biodiversity, understanding landscape features which underpin species diversity is crucial. Focusing on butterflies, we addressed this question for a cultural-historic landscape in Southern Transylvania, Romania. Following a natural experiment, we randomly selected 120 survey sites in farmland, 60 each in grassland and arable land. We surveyed butterfly species richness and abundance by walking transects with four repeats in summer 2012. We analysed species composition using Detrended Correspondence Analysis. We modelled species richness, richness of functional groups, and abundance of selected species in response to topography, woody vegetation cover and heterogeneity at three spatial scales, using generalised linear mixed effects models. Species composition widely overlapped in grassland and arable land. Composition changed along gradients of heterogeneity at local and context scales, and of woody vegetation cover at context and landscape scales. The effect of local heterogeneity on species richness was positive in arable land, but negative in grassland. Plant species richness, and structural and topographic conditions at multiple scales explained species richness, richness of functional groups and species abundances. Our study revealed high conservation value of both grassland and arable land in low-intensity Eastern European farmland. Besides grassland, also heterogeneous arable land provides important habitat for butterflies. While butterfly diversity in arable land benefits from heterogeneity by small-scale structures, grasslands should be protected from fragmentation to provide

  5. Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

    Science.gov (United States)

    Liu, Xing; Tang, Zongwen; Duan, Zhenhua; He, Zhenyun; Shu, Mei; Wang, Xianxian; Gee, Shirley J; Hammock, Bruce D; Xu, Yang

    2017-03-01

    A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC 50 of 0.64ng/mL and a linear range (IC 20 -IC 80 ) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. High-order conservative discretizations for some cases of the rigid body motion

    International Nuclear Information System (INIS)

    Kozlov, Roman

    2008-01-01

    Modified vector fields can be used to construct high-order structure-preserving numerical integrators for ordinary differential equations. In the present Letter we consider high-order integrators based on the implicit midpoint rule, which conserve quadratic first integrals. It is shown that these integrators are particularly suitable for the rigid body motion with an additional quadratic first integral. In this case high-order integrators preserve all four first integrals of motion. The approach is illustrated on the Lagrange top (a rotationally symmetric rigid body with a fixed point on the symmetry axis). The equations of motion are considered in the space fixed frame because in this frame Lagrange top admits a neat description. The Lagrange top motion includes the spherical pendulum and the planar pendulum, which swings in a vertical plane, as particular cases

  7. Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

    Science.gov (United States)

    Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

    2004-02-01

    We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

  8. High Sensitivity Electrochemical Cholesterol Sensor Utilizing a Vertically Aligned Carbon Nanotube Electrode with Electropolymerized Enzyme Immobilization

    Directory of Open Access Journals (Sweden)

    Ditsayut Phokharatkul

    2009-10-01

    Full Text Available In this report, a new cholesterol sensor is developed based on a vertically aligned CNT electrode with two-step electrochemical polymerized enzyme immobilization. Vertically aligned CNTs are selectively grown on a 1 mm2 window of gold coated SiO2/Si substrate by thermal chemical vapor deposition (CVD with gravity effect and water-assisted etching. CNTs are then simultaneously functionalized and enzyme immobilized by electrochemical polymerization of polyaniline and cholesterol enzymes. Subsequently, ineffective enzymes are removed and new enzymes are electrochemically recharged. Scanning electron microscopic characterization indicates polymer-enzyme nanoparticle coating on CNT surface. Cyclic voltammogram (CV measurements in cholesterol solution show the oxidation and reduction peaks centered around 450 and −220 mV, respectively. An approximately linear relationship between the cholesterol concentration and the response current could be observed in the concentration range of 50–300 mg/dl with a sensitivity of approximately 0.22 μA/mg·dl−1, which is considerably higher compared to previously reported CNT bioprobe. In addition, good specificity toward glucose, uric acid acetaminophen and ascorbic acid have been obtained. Moreover, sensors have satisfactory stability, repeatability and life time. Therefore, the electropolymerized CNT bioprobe is promising for cholesterol detection in normal cholesterol concentration in human blood.

  9. Traveling waves and conservation laws for highly nonlinear wave equations modeling Hertz chains

    Science.gov (United States)

    Przedborski, Michelle; Anco, Stephen C.

    2017-09-01

    A highly nonlinear, fourth-order wave equation that models the continuum theory of long wavelength pulses in weakly compressed, homogeneous, discrete chains with a general power-law contact interaction is studied. For this wave equation, all solitary wave solutions and all nonlinear periodic wave solutions, along with all conservation laws, are derived. The solutions are explicitly parameterized in terms of the asymptotic value of the wave amplitude in the case of solitary waves and the peak of the wave amplitude in the case of nonlinear periodic waves. All cases in which the solution expressions can be stated in an explicit analytic form using elementary functions are worked out. In these cases, explicit expressions for the total energy and total momentum for all solutions are obtained as well. The derivation of the solutions uses the conservation laws combined with an energy analysis argument to reduce the wave equation directly to a separable first-order differential equation that determines the wave amplitude in terms of the traveling wave variable. This method can be applied more generally to other highly nonlinear wave equations.

  10. Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event

    DEFF Research Database (Denmark)

    Danielsen, E M

    1992-01-01

    a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate...... enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces...

  11. High-throughput assay of 9 lysosomal enzymes for newborn screening.

    Science.gov (United States)

    Spacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C Ronald; Turecek, Frantisek; Gelb, Michael H

    2013-03-01

    There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

  12. Determining the drivers of population structure in a highly urbanized landscape to inform conservation planning.

    Science.gov (United States)

    Thomassen, Henri A; Harrigan, Ryan J; Semple Delaney, Kathleen; Riley, Seth P D; Serieys, Laurel E K; Pease, Katherine; Wayne, Robert K; Smith, Thomas B

    2018-02-01

    Understanding the environmental contributors to population structure is of paramount importance for conservation in urbanized environments. We used spatially explicit models to determine genetic population structure under current and future environmental conditions across a highly fragmented, human-dominated environment in Southern California to assess the effects of natural ecological variation and urbanization. We focused on 7 common species with diverse habitat requirements, home-range sizes, and dispersal abilities. We quantified the relative roles of potential barriers, including natural environmental characteristics and an anthropogenic barrier created by a major highway, in shaping genetic variation. The ability to predict genetic variation in our models differed among species: 11-81% of intraspecific genetic variation was explained by environmental variables. Although an anthropogenically induced barrier (a major highway) severely restricted gene flow and movement at broad scales for some species, genetic variation seemed to be primarily driven by natural environmental heterogeneity at a local level. Our results show how assessing environmentally associated variation for multiple species under current and future climate conditions can help identify priority regions for maximizing population persistence under environmental change in urbanized regions. © 2017 Society for Conservation Biology.

  13. A highly conserved tyrosine of Tim-3 is phosphorylated upon stimulation by its ligand galectin-9

    International Nuclear Information System (INIS)

    Weyer, Philipp S. van de; Muehlfeit, Michael; Klose, Christoph; Bonventre, Joseph V.; Walz, Gerd; Kuehn, E. Wolfgang

    2006-01-01

    Tim-3 is a member of the TIM family of proteins (T-cell immunoglobulin mucin) involved in the regulation of CD4+ T-cells. Tim-3 is a T H 1-specific type 1 membrane protein and regulates T H 1 proliferation and the development of tolerance. Binding of galectin-9 to the extracellular domain of Tim-3 results in apoptosis of T H 1 cells, but the intracellular pathways involved in the regulatory function of Tim-3 are unknown. Unlike Tim-1, which is expressed in renal epithelia and cancer, Tim-3 has not been described in cells other than neuronal or T-cells. Using RT-PCR we demonstrate that Tim-3 is expressed in malignant and non-malignant epithelial tissues. We have cloned Tim-3 from an immortalized liver cell carcinoma line and identified a highly conserved tyrosine in the intracellular tail of Tim-3 (Y265). We demonstrate that Y265 is specifically phosphorylated in vivo by the interleukin inducible T cell kinase (ITK), a kinase which is located in close proximity of the TIM genes on the allergy susceptibility locus 5q33.3. Stimulation of Tim-3 by its ligand galectin-9 results in increased phosphorylation of Y265, suggesting that this tyrosine residue plays an important role in downstream signalling events regulating T-cell fate. Given the role of TIM proteins in autoimmunity and cancer, the conserved SH2 binding domain surrounding Y265 could represent a possible target site for pharmacological intervention

  14. Effect of high hydrostatic pressure on mycelial development, spore viability and enzyme activity of Penicillium Roqueforti.

    Science.gov (United States)

    Martínez-Rodríguez, Yamile; Acosta-Muñiz, Carlos; Olivas, Guadalupe I; Guerrero-Beltrán, José; Rodrigo-Aliaga, Dolores; Mujica-Paz, Hugo; Welti-Chanes, Jorge; Sepulveda, David R

    2014-01-03

    This study investigated the effect of high hydrostatic pressure treatments on mycelial development, spore viability, and total proteolytic and lipolytic activity of Penicillium roqueforti PV-LYO 10 D. Fungus growing in liquid medium was pressure-treated at 300, 400, and 500 MPa for 10 min at 20°C following seven days of incubation at 25°C and analyzed periodically up to day 9 after treatments to evaluate the effect on fungal growth. Mycelial mass of P. roqueforti was significantly affected at all pressure treatments evaluated, being 15.48%, 22.28%, 30.03%, and 12.53% lower than controls on day 1, 3, 6, and 9 after 300 MPa treatment, respectively. In a similar way, at 400 and 500 MPa, mycelial mass was 31.08% and 60.34% lower than controls one day after treatments and 49.74% and 80.85% lower on day 9, respectively. The viability of P. roqueforti spores decreased by 36.53% at 300 MPa, and complete inactivation took place at ≥400 MPa from an initial count of 7 log cfu/mL. Total proteolytic activity was not significantly affected at 300 MPa but was reduced by 18.22% at 400 MPa and by 43.18% at 500 MPa. Total lipolytic activity also decreased as the intensity of the pressure treatments increased. 21.69%, 39.12%, and 56.26% activity reductions were observed when treatments of 300, 400 and 500 MPa were applied, respectively. The results from this study show that pressure treatments are able to control growth, inactivate spores, and alter enzyme activity of P. roqueforti, which could be of interest in extending the shelf-life of blue-veined cheeses and other food products. © 2013.

  15. An assessment of high carbon stock and high conservation value approaches to sustainable oil palm cultivation in Gabon

    Science.gov (United States)

    Austin, Kemen G.; Lee, Michelle E.; Clark, Connie; Forester, Brenna R.; Urban, Dean L.; White, Lee; Kasibhatla, Prasad S.; Poulsen, John R.

    2017-01-01

    Industrial-scale oil palm cultivation is rapidly expanding in Gabon, where it has the potential to drive economic growth, but also threatens forest, biodiversity and carbon resources. The Gabonese government is promoting an ambitious agricultural expansion strategy, while simultaneously committing to minimize negative environmental impacts of oil palm agriculture. This study estimates the extent and location of suitable land for oil palm cultivation in Gabon, based on an analysis of recent trends in plantation permitting. We use the resulting suitability map to evaluate two proposed approaches to minimizing negative environmental impacts: a High Carbon Stock (HCS) approach, which emphasizes forest protection and climate change mitigation, and a High Conservation Value (HCV) approach, which focuses on safeguarding biodiversity and ecosystems. We quantify the forest area, carbon stock, and biodiversity resources protected under each approach, using newly developed maps of priority species distributions and forest biomass for Gabon. We find 2.7-3.9 Mha of suitable or moderately suitable land that avoid HCS areas, 4.4 million hectares (Mha) that avoid HCV areas, and 1.2-1.7 Mha that avoid both. This suggests that Gabon’s oil palm production target could likely be met without compromising important ecosystem services, if appropriate safeguards are put in place. Our analysis improves understanding of suitability for oil palm in Gabon, determines how conservation strategies align with national targets for oil palm production, and informs national land use planning.

  16. GROWTH AND ENZYME PRODUCTION DURING CONTINUOUS CULTURES OF A HIGH AMYLASE-PRODUCING VARIANT OF Aspergillus Oryzae

    OpenAIRE

    Zangirolami,T.C.; Carlsen,M.; Nielsen,J.; Jørgensen,S.B.

    2002-01-01

    Growth and product formation by a selected variant of Aspergillus oryzae showing high alpha-amylase production was studied in continuous cultivations carried out at six different specific growth rates, using glucose as the growth-limiting nutrient. The analysis of the steady-state data revealed that the variant and wild-type strains were similar with respect to glucose uptake system and stoichiometric coefficients. However, the variant was capable of maintaining an enzyme production as high a...

  17. Highly active engineered-enzyme oriented monolayers: formation, characterization and sensing applications

    Directory of Open Access Journals (Sweden)

    Patolsky Fernando

    2011-06-01

    Full Text Available Abstract Background The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. Methods Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. Results and Discussion Water contact angle (65-70°, as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate and the other (SH/S-S at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution. Conclusions To the best of our knowledge, immobilization of a protein by the method

  18. Data on electrical energy conservation using high efficiency motors for the confidence bounds using statistical techniques.

    Science.gov (United States)

    Shaikh, Muhammad Mujtaba; Memon, Abdul Jabbar; Hussain, Manzoor

    2016-09-01

    In this article, we describe details of the data used in the research paper "Confidence bounds for energy conservation in electric motors: An economical solution using statistical techniques" [1]. The data presented in this paper is intended to show benefits of high efficiency electric motors over the standard efficiency motors of similar rating in the industrial sector of Pakistan. We explain how the data was collected and then processed by means of formulas to show cost effectiveness of energy efficient motors in terms of three important parameters: annual energy saving, cost saving and payback periods. This data can be further used to construct confidence bounds for the parameters using statistical techniques as described in [1].

  19. Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation

    DEFF Research Database (Denmark)

    Czaban, Adrian; Sharma, Sapna; Byrne, Stephen

    2015-01-01

    species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family...... clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity...... enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some...

  20. Cortical cytasters: a highly conserved developmental trait of Bilateria with similarities to Ctenophora

    Directory of Open Access Journals (Sweden)

    Salinas-Saavedra Miguel

    2011-12-01

    Full Text Available Abstract Background Cytasters (cytoplasmic asters are centriole-based nucleation centers of microtubule polymerization that are observable in large numbers in the cortical cytoplasm of the egg and zygote of bilaterian organisms. In both protostome and deuterostome taxa, cytasters have been described to develop during oogenesis from vesicles of nuclear membrane that move to the cortical cytoplasm. They become associated with several cytoplasmic components, and participate in the reorganization of cortical cytoplasm after fertilization, patterning the antero-posterior and dorso-ventral body axes. Presentation of the hypothesis The specific resemblances in the development of cytasters in both protostome and deuterostome taxa suggest that an independent evolutionary origin is unlikely. An assessment of published data confirms that cytasters are present in several protostome and deuterostome phyla, but are absent in the non-bilaterian phyla Cnidaria and Ctenophora. We hypothesize that cytasters evolved in the lineage leading to Bilateria and were already present in the most recent common ancestor shared by protostomes and deuterostomes. Thus, cytasters would be an ancient and highly conserved trait that is homologous across the different bilaterian phyla. The alternative possibility is homoplasy, that is cytasters have evolved independently in different lineages of Bilateria. Testing the hypothesis So far, available published information shows that appropriate observations have been made in eight different bilaterian phyla. All of them present cytasters. This is consistent with the hypothesis of homology and conservation. However, there are several important groups for which there are no currently available data. The hypothesis of homology predicts that cytasters should be present in these groups. Increasing the taxonomic sample using modern techniques uniformly will test for evolutionary patterns supporting homology, homoplasy, or secondary loss of

  1. Discovery of Microorganisms and Enzymes Involved in High-Solids Decomposition of Rice Straw Using Metagenomic Analyses

    Science.gov (United States)

    D’haeseleer, Patrik; Khudyakov, Jane; Burd, Helcio; Hadi, Masood; Simmons, Blake A.; Singer, Steven W.; Thelen, Michael P.; VanderGheynst, Jean S.

    2013-01-01

    High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production. PMID:24205054

  2. Discovery of microorganisms and enzymes involved in high-solids decomposition of rice straw using metagenomic analyses.

    Directory of Open Access Journals (Sweden)

    Amitha P Reddy

    Full Text Available High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C and thermophilic (55°C conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2 were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.

  3. Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Petranovic, D.; Købmann, Brian

    2010-01-01

    The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% ...

  4. Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes.

    Directory of Open Access Journals (Sweden)

    Luka Ausec

    Full Text Available Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three-domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.

  5. Lipase enzymes on graphene oxide support for high-efficiency biocatalysis

    Czech Academy of Sciences Publication Activity Database

    Hermanová, S.; Zarevúcka, Marie; Bouša, D.; Mikulics, M.; Sofer, Z.

    2016-01-01

    Roč. 5, Dec (2016), s. 200-208 ISSN 2352-9407 R&D Projects: GA ČR(CZ) GA15-09001S Institutional support: RVO:61388963 Keywords : biocatalysis * graphene oxide * enzyme * acylglycerols Subject RIV: CC - Organic Chemistry

  6. A highly conserved amino acid in VP1 regulates maturation of enterovirus 71.

    Directory of Open Access Journals (Sweden)

    Yong-Xin Zhang

    2017-09-01

    Full Text Available Enterovirus 71 (EV71 is the major causative agent of hand, foot and mouth disease (HFMD in children, causing severe clinical outcomes and even death. Here, we report an important role of the highly conserved alanine residue at position 107 in the capsid protein VP1 (VP1A107 in the efficient replication of EV71. Substitutional mutations of VP1A107 significantly diminish viral growth kinetics without significant effect on viral entry, expression of viral genes and viral production. The results of mechanistic studies reveal that VP1A107 regulates the efficient cleavage of the VP0 precursor during EV71 assembly, which is required, in the next round of infection, for the transformation of the mature virion (160S into an intermediate or A-particle (135S, a key step of virus uncoating. Furthermore, the results of molecular dynamic simulations and hydrogen-bond networks analysis of VP1A107 suggest that flexibility of the VP1 BC loop or the region surrounding the VP1107 residue directly correlates with viral infectivity. It is possible that sufficient flexibility of the region surrounding the VP1107 residue favors VP0 conformational change that is required for the efficient cleavage of VP0 as well as subsequent viral uncoating and viral replication. Taken together, our data reveal the structural role of the highly conserved VP1A107 in regulating EV71 maturation. Characterization of this novel determinant of EV71 virulence would promote the study on pathogenesis of Enteroviruses.

  7. CpG methylation differences between neurons and glia are highly conserved from mouse to human.

    Science.gov (United States)

    Kessler, Noah J; Van Baak, Timothy E; Baker, Maria S; Laritsky, Eleonora; Coarfa, Cristian; Waterland, Robert A

    2016-01-15

    Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type-specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non-CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type-specific differences in CpG methylation, with a genome-wide tendency for neuronal CpG-hypermethylation punctuated by regions of glia-specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type-specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene-associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome-wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Scharfe Maren

    2010-04-01

    Full Text Available Abstract Background The constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. New sequencing technologies are now on the horizon that will yield massive increases in our capacity for DNA sequencing and will revolutionize the drug discovery process. Since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus. Results In this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than 10 individual isolates. Pooled sequencing of 36 clinical Pseudomonas aeruginosa isolates revealed that essential and highly expressed proteins evolve at lower rates, whereas extracellular proteins evolve at higher rates. We furthermore refined the list of experimentally essential P. aeruginosa genes, and identified 980 genes that show no sequence variation at all. Among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen. Conclusion The detailed analysis of a comprehensive set of P. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. Sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies.

  9. A guide to calculating habitat-quality metrics to inform conservation of highly mobile species

    Science.gov (United States)

    Bieri, Joanna A.; Sample, Christine; Thogmartin, Wayne E.; Diffendorfer, James E.; Earl, Julia E.; Erickson, Richard A.; Federico, Paula; Flockhart, D. T. Tyler; Nicol, Sam; Semmens, Darius J.; Skraber, T.; Wiederholt, Ruscena; Mattsson, Brady J.

    2018-01-01

    Many metrics exist for quantifying the relative value of habitats and pathways used by highly mobile species. Properly selecting and applying such metrics requires substantial background in mathematics and understanding the relevant management arena. To address this multidimensional challenge, we demonstrate and compare three measurements of habitat quality: graph-, occupancy-, and demographic-based metrics. Each metric provides insights into system dynamics, at the expense of increasing amounts and complexity of data and models. Our descriptions and comparisons of diverse habitat-quality metrics provide means for practitioners to overcome the modeling challenges associated with management or conservation of such highly mobile species. Whereas previous guidance for applying habitat-quality metrics has been scattered in diversified tracks of literature, we have brought this information together into an approachable format including accessible descriptions and a modeling case study for a typical example that conservation professionals can adapt for their own decision contexts and focal populations.Considerations for Resource ManagersManagement objectives, proposed actions, data availability and quality, and model assumptions are all relevant considerations when applying and interpreting habitat-quality metrics.Graph-based metrics answer questions related to habitat centrality and connectivity, are suitable for populations with any movement pattern, quantify basic spatial and temporal patterns of occupancy and movement, and require the least data.Occupancy-based metrics answer questions about likelihood of persistence or colonization, are suitable for populations that undergo localized extinctions, quantify spatial and temporal patterns of occupancy and movement, and require a moderate amount of data.Demographic-based metrics answer questions about relative or absolute population size, are suitable for populations with any movement pattern, quantify demographic

  10. Habitat Re-Creation (Ecological Restoration) as a Strategy for Conserving Insect Communities in Highly Fragmented Landscapes.

    Science.gov (United States)

    Shuey, John A

    2013-12-05

    Because of their vast diversity, measured by both numbers of species as well as life history traits, insects defy comprehensive conservation planning. Thus, almost all insect conservation efforts target individual species. However, serious insect conservation requires goals that are set at the faunal level and conservation success requires strategies that conserve intact communities. This task is complicated in agricultural landscapes by high levels of habitat fragmentation and isolation. In many regions, once widespread insect communities are now functionally trapped on islands of ecosystem remnants and subject to a variety of stressors associated with isolation, small population sizes and artificial population fragmentation. In fragmented landscapes ecological restoration can be an effective strategy for reducing localized insect extinction rates, but insects are seldom included in restoration design criteria. It is possible to incorporate a few simple conservation criteria into restoration designs that enhance impacts to entire insect communities. Restoration can be used as a strategy to address fragmentation threats to isolated insect communities if insect communities are incorporated at the onset of restoration planning. Fully incorporating insect communities into restoration designs may increase the cost of restoration two- to three-fold, but the benefits to biodiversity conservation and the ecological services provided by intact insect communities justify the cost.

  11. Butterflies of the high altitude Atacama Desert: habitat use and conservation

    Directory of Open Access Journals (Sweden)

    Emma eDespland

    2014-09-01

    Full Text Available The butterfly fauna of the high-altitude desert of Northern Chile, though depauperate, shows high endemism, is poorly known and is of considerable conservation concern. This study surveys butterflies along the Andean slope between 2400 and 500 m asl (prepuna, puna and Andean steppe habitats as well as in high and low altitude wetlands and in the neoriparian vegetation of agricultural sites. We also include historical sightings from museum records. We compare abundances between altitudes, between natural and impacted sites, as well as between two sampling years with different precipitation regimes. The results confirm high altitudinal turnover and show greatest similarity between wetland and slope faunas at similar altitudes. Results also underscore vulnerability to weather fluctuations, particularly in the more arid low-altitude sites, where abundances were much lower in the low precipitation sampling season and several species were not observed at all. Finally, we show that some species have shifted to the neoriparian vegetation of the agricultural landscape, whereas others were only observed in less impacted habitats dominated by native plants. These results suggest that acclimation to novel habitats depends on larval host plant use. The traditional agricultural environment can provide habitat for many, but not all, native butterfly species, but an estimation of the value of these habitats requires better understanding of butterfly life-history strategies and relationships with host plants.

  12. Angiotensin-converting enzyme genotype and arterial oxygen saturation at high altitude in Peruvian Quechua.

    Science.gov (United States)

    Bigham, Abigail W; Kiyamu, Melisa; León-Velarde, Fabiola; Parra, Esteban J; Rivera-Ch, Maria; Shriver, Mark D; Brutsaert, Tom D

    2008-01-01

    The I-allele of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism has been associated with performance benefits at high altitude (HA). In n = 142 young males and females of largely Quechua origins in Peru, we evaluated 3 specific hypotheses with regard to the HA benefits of the I-allele: (1) the I-allele is associated with higher arterial oxygen saturation (Sa(O(2))) at HA, (2) the I-allele effect depends on the acclimatization state of the subjects, and (3) the putative I-allele effect on Sa(O(2)) is mediated by the isocapnic hypoxic ventilatory response (HVR, l/min(1)/% Sa(O(2))(1)). The subject participants comprised two different study groups including BLA subjects (born at low altitude) who were lifelong sea-level residents transiently exposed to hypobaric hypoxia (<24 h) and BHA subjects (born at HA) who were lifelong residents of HA. To control for the possibility of population stratification, Native American ancestry proportion (NAAP) was estimated as a covariate for each individual using a panel of 70 ancestry-informative molecular markers (AIMS). At HA, resting and exercise Sa(O(2)) was strongly associated with the ACE genotype, p = 0.008 with approximately 4% of the total variance in Sa(O(2)) attributed to ACE genotype. Moreover, I/I individuals maintained approximately 2.3 percentage point higher Sa(O(2)) compared to I/D and D/D. This I-allele effect was evident in both BLA and BHA groups, suggesting that acclimatization state has little influence on the phenotypic expression of the ACE gene. Finally, ACE genotype was not associated with the isocapnic HVR, although HVR had a strong independent effect on Sa(O(2)) (p = 0.001). This suggests that the I-allele effect on Sa(O(2)) is not mediated by the peripheral control of breathing, but rather by some other central cardiopulmonary effect of the ACE gene on the renin-angiotensin-aldosterone system (RAAS).

  13. Cosmetic results in early stage breast cancer patients with high-dose brachytherapy after conservative surgery

    International Nuclear Information System (INIS)

    Torres, Felipe; Pineda, Beatriz E

    2004-01-01

    Purpose: to reveal cosmetic results in patients at early stages of low risk breast cancer treated with partial accelerated radiotherapy using high dose rate brachytherapy. Methods and materials: from March 2001 to July 2003,14 stages l and ll breast cancer patients were treated at the Colombian national cancer institute in Bogota with conservative surgery and radiotherapy upon the tumor bed (partial accelerated radiotherapy), using interstitial implants with iridium 192 (high dose rate brachytherapy) with a dose of 32 Gys, over 4 days, at 8 fractions twice a day. Results: with an average follow up of 17.7 months, good cosmetic results were found among 71.4 % of patients and excellent results among 14.3% of patients, furthermore none of the patients neither local nor regional or distant relapses. Conclusion: among patients who suffer from breast cancer at early stages, it showed is possible to apply partial accelerated radiotherapy upon the tumor bed with high doses over 4 days with good to excellent cosmetic results

  14. Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?

    DEFF Research Database (Denmark)

    Cannella, David; Jørgensen, Henning

    2014-01-01

    proven essential for economic feasibility at industrial scale. Historically, simultaneous saccharification and fermentation (SSF) was found to give better ethanol yields compared to separate hydrolysis and fermentation (SHF), but data in literature are typically based on operating the process at low dry...... matter conditions. In this work the impact of selected enzyme preparation and processing strategy (SHF, presaccharification and simultaneous saccharification and fermentation—PSSF, and SSF) on final ethanol yield and overall performance was investigated with pretreated wheat straw up to 30% DM...... cellulose to around 94%, revealing that the most relevant products could be accounted for. One observation was the presence of oxidized sugar (gluconic acid) upon enzymatic hydrolysis with the latest enzyme preparation. Experiments showed gluconic acid formation by recently discovered enzymatic class...

  15. The Teaching of Enzymes: A Low Cost Experimental Approach with High School Students.

    Directory of Open Access Journals (Sweden)

    W. A PINHEIRO

    2009-05-01

    Full Text Available The association of experimental methods with the traditional lecturing of Science themes is encouraged by several authors. The importance ofconducting experimental classes is undeniable, as practical activities motivate students to search for kno wledge. But this is not the reality in most publicschools in the inlands of Rio de Janeiro’s State, where several factors prevent teachers from using such didactic strategy.   The aim of this  work  was demonstrate  low -cost experimental activities, addressing the subject enzymes. The practice has been designed for being held in a common classroom. Lab glassware was replaced by alternative materials. Potato extract  (catalase source and commercial hydrogen peroxide were required. Concepts such as kinetic data  related to substrate concentration, time of reaction, pH andtemperature effects on enzymatic activity have been  explored with the students. Graphical representation of enzyme activity related to the above parameters has also been elaborated. The practice w as held in 10 schools distributed among 7 different cities. Questionnaires have been applied to the students before and after the practice and revealed that 86% of the students had never participated of a practical lesson and 49% were unaware of the functi on of enzymes. After practice, 68% of the students said that the enzymes catalyze biological reactions and 73% considered the practice important to their cognitive achievement. The evaluation of information obtained from students confirmed the lack of practical activities in public education, as well as the capacity that the practical activities have  to motivate the students in the knowledge formation process.

  16. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    International Nuclear Information System (INIS)

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

  17. Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening.

    Science.gov (United States)

    Lee, Moo-Yeal; Dordick, Jonathan S; Clark, Douglas S

    2010-01-01

    Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To accelerate the identification of safer drug candidates and improve the clinical progression of drug candidates to pharmaceuticals, it is important to develop high-throughput tools that can provide early-stage predictive toxicology data. In particular, in vitro cell-based systems that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process. The in vitro techniques that provide a high degree of human toxicity prediction will be perhaps more important in cosmetic and chemical industries in Europe, as animal toxicity testing is being phased out entirely in the immediate future.We have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized three-dimensional (3D) cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products. The human or rat MetaChip contains an array of encapsulated metabolic enzymes that is designed to emulate the metabolic reactions in the human or rat liver. The human or rat DataChip contains an array of 3D human or rat cells encapsulated in alginate gels for cell-based toxicity screening. By combining the DataChip with the complementary MetaChip, in vitro toxicity results are obtained that correlate well with in vivo rat data.

  18. Pancreatic Enzymes

    Science.gov (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  19. Swertia chirayta, a Threatened High-Value Medicinal Herb: Microhabitats and Conservation Challenges in Sikkim Himalaya, India

    Directory of Open Access Journals (Sweden)

    Bharat Kumar Pradhan

    2015-11-01

    Full Text Available Assessing the impact of threats, identifying favorable growing conditions, and predicting future population scenarios are vital for the conservation and management of threatened species. This study investigated the availability, microhabitat characteristics, threat status, and community associations of Swertia chirayta, a highly threatened Himalayan medicinal herb, in 22 populations in Sikkim, India, using the vertical belt transect method. Of the 14 microhabitats identified, open grassy slope emerged as the most favorable and wet grassy slope as the least favorable for S. chirayta. The species was dominant in 8 of the 10 major plant communities identified. Among 9 major types of disturbance identified, human movement and collection of non-timber forest products appeared as the biggest threats to S. chirayta. Disturbances significantly affected the availability of the species. S. chirayta, though under high anthropogenic threat, maintains high microhabitat pliability, which is vital for its conservation and management, provided immediate conservation measures are taken.

  20. Co-solvent pretreatment reduces costly enzyme requirements for high sugar and ethanol yields from lignocellulosic biomass.

    Science.gov (United States)

    Nguyen, Thanh Yen; Cai, Charles M; Kumar, Rajeev; Wyman, Charles E

    2015-05-22

    We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Evaluation of highly conserved hsp65-specific nested PCR primers for diagnosing Mycobacterium tuberculosis.

    Science.gov (United States)

    Priyadarshini, P; Tiwari, K; Das, A; Kumar, D; Mishra, M N; Desikan, P; Nath, G

    2017-02-01

    To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65). The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers. The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg. The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.

  2. Enzymatic hydrolysis and ethanol fermentation of high dry matter wet-exploded wheat straw at low enzyme loading

    DEFF Research Database (Denmark)

    Georgieva, T.I.; Hou, Xiaoru; Hilstrøm, Troels

    2008-01-01

    was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15-20% will be necessary. However, high DM hydrolysis and fermentation are limited by high...... and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%....

  3. Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes.

    Directory of Open Access Journals (Sweden)

    Benjamin R Jack

    2016-05-01

    Full Text Available Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein-protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes.

  4. Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

    OpenAIRE

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.; Papke, Roger L.

    2009-01-01

    We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (α7 Tyr188 or α4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the α7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in α4β2 and α7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human α7 and α4β2 and expressed the receptors in Xenopus laevis oocytes. α7 Re...

  5. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    Science.gov (United States)

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  6. High titers of autoantibodies to glutamate decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

    Science.gov (United States)

    Hampe, Christiane S.; Maitland, Murray E.; Gilliam, Lisa K.; Thi Phan, Thanh-H.; Sweet, Ian R.; Radtke, Jared R.; Bota, Vasile; Ransom, Bruce R.; Hirsch, Irl B.

    2014-01-01

    Objective Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders and patients with type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesize that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. Methods We analyzed GAD65Ab titer, inhibition of GAD65 enzyme activity, and pattern of GAD65Ab epitopes in a cohort of type 1 diabetes patients (n=100) and correlated our findings with neurological symptoms and diseases. Results Fourty-three percent (43/100) of the patients had detectable GAD65Ab titers (median=400 U/ml, range: 142–250,000U/ml). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000–250,000 U/ml). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of five patients inhibited the enzyme activity significantly (by 34–55%). Three of these patients complained of muscle stiffness and pain, which was documented in two of these patients. Conclusions Based on our findings we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. PMID:23512385

  7. Production of Biodiesel from High Acid Value Waste Cooking Oil Using an Optimized Lipase Enzyme/Acid-Catalyzed Hybrid Process

    Directory of Open Access Journals (Sweden)

    N. Saifuddin

    2009-01-01

    Full Text Available The present study is aimed at developing an enzymatic/acid-catalyzed hybrid process for biodiesel production using waste cooking oil with high acid value (poor quality as feedstock. Tuned enzyme was prepared using a rapid drying technique of microwave dehydration (time required around 15 minutes. Further enhancement was achieved by three phase partitioning (TPP method. The results on the lipase enzyme which was subjected to pH tuning and TPP, indicated remarkable increase in the initial rate of transesterification by 3.8 times. Microwave irradiation was found to increase the initial reaction rates by further 1.6 times, hence giving a combined increase in activity of about 5.4 times. The optimized enzyme was used for hydrolysis and 88% of the oil taken initially was hydrolyzed by the lipase. The hydrolysate was further used in acid-catalyzed esterification for biodiesel production. By using a feedstock to methanol molar ratio of 1:15 and a sulphuric acid concentration of 2.5%, a biodiesel conversion of 88% was obtained at 50 °C for an hour reaction time. This hybrid process may open a way for biodiesel production using unrefined and used oil with high acid value as feedstock.

  8. Acacia shrubs respond positively to high severity wildfire: Implications for conservation and fuel hazard management.

    Science.gov (United States)

    Gordon, Christopher E; Price, Owen F; Tasker, Elizabeth M; Denham, Andrew J

    2017-01-01

    High severity wildfires pose threats to human assets, but are also perceived to impact vegetation communities because a small number of species may become dominant immediately after fire. However there are considerable gaps in our knowledge about species-specific responses of plants to different fire severities, and how this influences fuel hazard in the short and long-term. Here we conduct a floristic survey at sites before and two years after a wildfire of unprecedented size and severity in the Warrumbungle National Park (Australia) to explore relationships between post-fire growth of a fire responsive shrub genera (Acacia), total mid-story vegetation cover, fire severity and fuel hazard. We then survey 129 plots surrounding the park to assess relationships between mid-story vegetation cover and time-since-fire. Acacia species richness and cover were 2.3 and 4.3 times greater at plots after than before the fire. However the same common dominant species were present throughout the study. Mid-story vegetation cover was 1.5 times greater after than before the wildfire, and Acacia species contribution to mid-story cover increased from 10 to 40%. Acacia species richness was not affected by fire severity, however strong positive associations were observed between Acacia and total mid-story vegetation cover and severity. Our analysis of mid-story vegetation recovery showed that cover was similarly high between 2 and 30years post-fire, then decreased until 52years. Collectively, our results suggest that Acacia species are extremely resilient to high severity wildfire and drive short to mid-term increases in fuel hazard. Our results are discussed in relation to fire regime management from the twin perspectives of conserving biodiversity and mitigating human losses due to wildfire. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries.

    Science.gov (United States)

    Sana, Barindra; Chia, Kuan Hui Burton; Raghavan, Sarada S; Ramalingam, Balamurugan; Nagarajan, Niranjan; Seayad, Jayasree; Ghadessy, Farid J

    2017-01-01

    Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available. We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals. We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that

  10. A dominant EV71-specific CD4+ T cell epitope is highly conserved among human enteroviruses.

    Directory of Open Access Journals (Sweden)

    Ruicheng Wei

    Full Text Available CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD, has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.

  11. Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes.

    Science.gov (United States)

    Srinivasachary; Dida, Mathews M; Gale, Mike D; Devos, Katrien M

    2007-08-01

    Finger millet is an allotetraploid (2n = 4x = 36) grass that belongs to the Chloridoideae subfamily. A comparative analysis has been carried out to determine the relationship of the finger millet genome with that of rice. Six of the nine finger millet homoeologous groups corresponded to a single rice chromosome each. Each of the remaining three finger millet groups were orthologous to two rice chromosomes, and in all the three cases one rice chromosome was inserted into the centromeric region of a second rice chromosome to give the finger millet chromosomal configuration. All observed rearrangements were, among the grasses, unique to finger millet and, possibly, the Chloridoideae subfamily. Gene orders between rice and finger millet were highly conserved, with rearrangements being limited largely to single marker transpositions and small putative inversions encompassing at most three markers. Only some 10% of markers mapped to non-syntenic positions in rice and finger millet and the majority of these were located in the distal 14% of chromosome arms, supporting a possible correlation between recombination and sequence evolution as has previously been observed in wheat. A comparison of the organization of finger millet, Panicoideae and Pooideae genomes relative to rice allowed us to infer putative ancestral chromosome configurations in the grasses.

  12. A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

    Science.gov (United States)

    Sanz-Martín, José M; Pacheco-Arjona, José Ramón; Bello-Rico, Víctor; Vargas, Walter A; Monod, Michel; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-09-01

    Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling. © 2015 BSPP and John Wiley & Sons Ltd.

  13. Pig epidermal growth factor precursor contains segments that are highly conserved among species

    DEFF Research Database (Denmark)

    Jørgensen, P E; Jensen, L.G.; Sørensen, B S

    1998-01-01

    segment with that of the human, the rat and the mouse EGF precursors, in order to identify highly conserved domains. The examined part of the precursor contains EGF itself and six so-called EGF-like modules. The overall amino acid identity among the four species is 64%. However, the amino acid identity...... differed from around 30% in some segments to around 70% in others. The highest amino acid identity, 71%, was observed for a 345-aa segment that contains three EGF-like modules and which is homologous to a part of the low-density lipoprotein receptor (LDL receptor). The amino acid identities are 64% for EGF...... itself, and 50-67% for the remaining three EGF-like modules. The segment of the LDL receptor that is homologous to a part of the EGF precursor is important for the function of the LDL receptor, and EGF-like modules seem to be involved in protein-protein interactions in a number of proteins. In conclusion...

  14. Current situation of energy conservation in high energy-consuming industries in Taiwan

    International Nuclear Information System (INIS)

    Chan, D.Y.-L.; Yang, K.-H.; Hsu, C.-H.; Chien, M.-H.; Hong, G.-B.

    2007-01-01

    Growing concern in Taiwan has arisen about energy consumption and its adverse environmental impact. The current situation of energy conservation in high energy-consuming industries in Taiwan, including the iron and steel, chemical, cement, pulp and paper, textiles and electric/electrical industries has been presented. Since the energy consumption of the top 100 energy users (T100) comprised over 50% of total industry energy consumption, focusing energy consumption reduction efforts on T100 energy users can achieve significant results. This study conducted on-site energy audits of 314 firms in Taiwan during 2000-2004, and identified potential electricity savings of 1,022,656 MWH, fuel oil savings of 174,643 kiloliters (KL), steam coal savings of 98,620 ton, and natural gas (NG) savings of 10,430 kilo cubic meters. The total potential energy saving thus was 489,505 KL of crude oil equivalent (KLOE), representing a reduction of 1,447,841 ton in the carbon dioxide emissions, equivalent to the annual carbon dioxide absorption capacity of a 39,131-ha plantation forest

  15. Effect of Bacillus cereus Enzymes on Milk Quality following Ultra High Temperature Processing

    Directory of Open Access Journals (Sweden)

    B. Janštová

    2006-01-01

    Full Text Available Using a model case of contamination of long-life semi-skimmed milk with the spores of six B. cereus strains, isolated from the farm environment and raw milk, proteolysis was monitored by measuring changes in protein content by infra-red spectroscopy; free tyrosine was measured by the Lowry method according to Juffs, and the reduction in casein fractions by SDS-PAGE. Lipolysis was monitored by the dilution extractive method. At a storage temperature of 4 °C for 4 months no enzyme processes were observed, whereas at a storage temperature of 24 °C a marked enzyme activity was found during maximum 3 weeks as well as sensory changes of UHT milk. After three weeks of storage, a reduction in protein content from 34.55 g l-1 milk to 29.46 ± 2.00 g l-1 milk, and a reduction in the free tyrosine from 0.65 to 2.13 ± 0.28 mg ml-1 was found, as well as increased molar contents of free fatty acids (FFA from 41.97 to 1617.22 ± 68.17 mmol kg-1 milk fat. After six days of storage, α-casein, β-casein and κ-casein dropped to 69 ± 10%, 56 ± 16% and 43 ± 10%, respectively. Majority of changes in UHT milk depended on the B. cereus strain used, initial microbial counts and the method of heat inactivation of spores.

  16. Biological pretreatment of corn stover with ligninolytic enzyme for high efficient enzymatic hydrolysis.

    Science.gov (United States)

    Wang, Feng-Qin; Xie, Hui; Chen, Wei; Wang, En-Tao; Du, Feng-Guang; Song, An-Dong

    2013-09-01

    Aiming at increasing the efficiency of transferring corn stover into sugars, a biological pretreatment was developed and investigated in this study. The protocol was characterized by the pretreatment with crude ligninolytic enzymes from Phanerochete chrysosporium and Coridus versicolor to break the lignin structure in corn stover, followed by a washing procedure to eliminate the inhibition of ligninolytic enzyme on cellulase. By a 2 d-pretreatment, sugar yield from corn stover hydrolysis could be increased by 50.2% (up to 323 mg/g) compared with that of the control. X-ray diffractometry and FT-IR analysis revealed that biological pretreatment could partially remove the lignin of corn stover, and consequently enhance the enzymatic hydrolysis efficiency of cellulose and hemeicellulose. In addition, the amount of microbial inhibitors, such as acetic acid and furfural, were much lower in biological pretreatment than that in acid pretreatment. This study provided a promising pretreatment method for biotransformation of corn stovers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    Energy Technology Data Exchange (ETDEWEB)

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  18. Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy.

    Science.gov (United States)

    Alzahrani, Khalid J H; Ali, Juma A M; Eze, Anthonius A; Looi, Wan Limm; Tagoe, Daniel N A; Creek, Darren J; Barrett, Michael P; de Koning, Harry P

    2017-08-01

    Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [ 3 H]-uridine but not of [ 3 H]-inosine. Conversely, the NT2-like genes mediated uptake of [ 3 H]-inosine but not [ 3 H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights

  19. Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy

    Directory of Open Access Journals (Sweden)

    Khalid J.H. Alzahrani

    2017-08-01

    Full Text Available Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU and 5F,2′deoxyuridine (5F,2′dUrd, and was designated uridine-uracil transporter 1 (UUT1; the other transporter mediated uptake of adenosine, uridine, 5F,2′dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1. To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2′dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2′dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy. Keywords: Leishmania, Pyrimidine metabolism, Uracil

  20. High precision conformal radiotherapy employing conservative margins in childhood benign and low-grade brain tumours

    International Nuclear Information System (INIS)

    Jalali, Rakesh; Budrukkar, Ashwini; Sarin, Rajiv; Sharma, Dayananda S.

    2005-01-01

    Background and purpose: To report local control and follow up outcome data of high precision conformal radiotherapy in childhood brain tumours. Materials and methods: Between December 1999 and December 2002, 26 children (17 boys and 9 girls, median age 11.5 years) with incompletely excised or recurrent benign and low-grade brain tumours [13 craniopharyngiomas, 11 low-grade gliomas (LGG) and 2 others] were treated with three-dimensional (3D) conformal radiotherapy (CRT) (12 patients) and stereotactic conformal radiotherapy (SCRT) (14 patients). Gross tumour volume (GTV) included neuro-imaging based visible tumour and/or resected tumour bed. Clinical target volume (CTV) consisted of GTV + 5 mm margin and planning target volume (PTV) consisted of additional 5 mm margin for CRT and 2 mm for SCRT. Treatment was delivered with 3-9 conformal fixed fields to a median dose of 54 Gy/30 fractions. Results: The actuarial 2 and 3 year disease free and overall survival was 96 and 100%, respectively (median follow up: 25 months, range 12-47 months). Radiological follow up available in 25 patients revealed complete response in 1, partial regression in 10, stable disease in 13 and progression in 1 patient (within the CTV). One patient with craniopharyngioma on a routine imaging revealed a mild asymptomatic cyst enlargement, which resolved with conservative management. A patient with chiasmatic glioma developed cystic degeneration and hydrocephalus 9 months after SCRT requiring cyst drainage and placement of a ventriculoperitoneal shunt. Conclusion: High-precision conformal techniques delivering irradiation to a computer generated target volume employing 7-10 mm 3D margins beyond the visible tumour and/or resected tumour bed appear to be safe in children with incompletely resected or recurrent benign and low-grade brain tumours, based on these data

  1. Discovering an Accessible Enzyme: Salivary [alpha]-Amylase--"Prima Digestio Fit in Ore"--A Didactic Approach for High School Students

    Science.gov (United States)

    Marini, Isabella

    2005-01-01

    Human salivary [alpha]-amylase is used in this experimental approach to introduce biology high school students to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then in a semi-quantitative…

  2. Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

    Energy Technology Data Exchange (ETDEWEB)

    Saddler, J N

    1982-11-01

    Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase (1,4-(1,3;1,4)-beta-D-glucan 4- glucanohydrolase, EC 3.2.1.4) activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) activity was approximately six to nine times greater. (Refs. 26).

  3. Isolation and screening of strains producing high amounts of rutin degrading enzymes from Fagopyrum tataricum seeds.

    Science.gov (United States)

    Zheng, Ya-Di; Luo, Qing-Lin; Zhou, Mei-Liang; Wang, De-Zhou; Zhang, Ye-Dong; Shao, Ji-Rong; Zhu, Xue-Mei; Tang, Yu

    2013-02-01

    The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation.

    Science.gov (United States)

    O'Brien, Aileen B; Philp, Kevin; Morris, Edwin R

    2009-09-28

    Cohesive gels have been obtained by de-esterification of 1.0wt% high-methoxy citrus pectin (degree of esterification approximately 68%) in the presence of Ca(2+) cations, using a commercial preparation (NovoShape) of fungal methyl esterase cloned from Aspergillus aculeatus. A convenient rate of network formation (gelation within approximately 30min) was achieved at an enzyme concentration of 0.2 PEU/g pectin. At a Ca(2+)-concentration of 40mM and incubation temperature of 20 degrees C, severe syneresis (>7% of sample mass) was observed, but release of fluid decreased with decreasing concentration of Ca(2+) and increasing temperature of incubation, becoming undetectable for 10mM Ca(2+) at 30 degrees C. Under these conditions, progressive development of solid-like character (storage modulus, G') was observed during 160min of enzymic de-esterification, and the mechanical spectrum recorded at the end of the incubation period had the form typical of a biopolymer gel. On subsequent heating to 70 degrees C, dissociation of the gel network (sigmoidal reduction in G' and G'') was observed. At or above the midpoint temperature of this melting process ( approximately 50 degrees C), there was no indication of gel formation on enzymic de-esterification (at 50 or 60 degrees C). At lower temperatures (20, 30 and 40 degrees C), the rate of gelation (assessed visually) showed no systematic increase as the incubation temperature was increased towards the temperature-optimum of the enzyme ( approximately 50 degrees C). This unexpected behaviour is attributed to competition between faster de-esterification and slower formation of Ca(2+)-induced 'egg-box' junctions.

  5. The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion

    OpenAIRE

    Schröter, Christina; Klupp, Barbara G.; Fuchs, Walter; Gerhard, Marika; Backovic, Marija; Rey, Felix A.; Mettenleiter, Thomas C.

    2014-01-01

    Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure sho...

  6. RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

    Science.gov (United States)

    Forest, David; Nishikawa, Ryuhei; Kobayashi, Hiroshi; Parton, Angela; Bayne, Christopher J.; Barnes, David W.

    2007-01-01

    We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3′ UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3′ mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology. PMID:17227856

  7. Gains of ubiquitylation sites in highly conserved proteins in the human lineage

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.

  8. High risks of losing genetic diversity in an endemic Mauritian gecko: implications for conservation.

    Directory of Open Access Journals (Sweden)

    Steeves Buckland

    Full Text Available Genetic structure can be a consequence of recent population fragmentation and isolation, or a remnant of historical localised adaptation. This poses a challenge for conservationists since misinterpreting patterns of genetic structure may lead to inappropriate management. Of 17 species of reptile originally found in Mauritius, only five survive on the main island. One of these, Phelsuma guimbeaui (lowland forest day gecko, is now restricted to 30 small isolated subpopulations following severe forest fragmentation and isolation due to human colonisation. We used 20 microsatellites in ten subpopulations and two mitochondrial DNA (mtDNA markers in 13 subpopulations to: (i assess genetic diversity, population structure and genetic differentiation of subpopulations; (ii estimate effective population sizes and migration rates of subpopulations; and (iii examine the phylogenetic relationships of haplotypes found in different subpopulations. Microsatellite data revealed significant population structure with high levels of genetic diversity and isolation by distance, substantial genetic differentiation and no migration between most subpopulations. MtDNA, however, showed no evidence of population structure, indicating that there was once a genetically panmictic population. Effective population sizes of ten subpopulations, based on microsatellite markers, were small, ranging from 44 to 167. Simulations suggested that the chance of survival and allelic diversity of some subpopulations will decrease dramatically over the next 50 years if no migration occurs. Our DNA-based evidence reveals an urgent need for a management plan for the conservation of P. guimbeaui. We identified 18 threatened and 12 viable subpopulations and discuss a range of management options that include translocation of threatened subpopulations to retain maximum allelic diversity, and habitat restoration and assisted migration to decrease genetic erosion and inbreeding for the viable

  9. The existence of High Conservation Value Forest (HCVF in Perum Perhutani KPH Kendal to support Implementation of FSC Certification

    Directory of Open Access Journals (Sweden)

    Sulistyowati Sri

    2018-01-01

    Full Text Available High Conservation Value Forest (HCVF is the identification of High Conservation Values that are important and need to be protected. Under FSC certification mechanism, HCVF becomes one of Principles and Criteria to attain certification. In this study, we identify the existence of HCVF in Perum Perhutani KPH Kendal to support implementation process of FSC certification. Qualitative method was conducted through observation and secondary data from Perum Perhutani KPH Kendal. Data analysis showed through ecolabel certification, Perum Perhutani KPH Kendal has been identified HCVF area covering 2,715.5 hectares consists of HCV 1 until 6. Secondary Natural Forest (HAS Subah and Kaliwungu for Ulolanang and Pagerwunung Nature Reserve buffer zone include as HCV 1.1, conservation area of leopard (Panthera pardus melas and Pangolin (Manis javanica.for HCV 1.2, conservation area of lutung (Trachypiyhecus auratus as endemic species for CITES App I and Critically Endangered species include as HCV 1.3, Goa kiskendo for bats species habitat include as HCV 1.4, regions of interest species for Deer (Cervus timorensis and Kepodang (Oriolus chinensis as HCV 2.3, Germplasm Protection Region/ KPPN area with high biodiversity include as HCV 3, river border area and water springs for HCV 4. While, utilization of firewood, grass for cattle fodder include as HCV 5 and 14 cultural sites include as HCV 6. From monitoring and evaluation of HCVF data, showed that in 2011-2015 the level of diversity for flora and fauna were increased.

  10. The existence of High Conservation Value Forest (HCVF) in Perum Perhutani KPH Kendal to support Implementation of FSC Certification

    Science.gov (United States)

    Sulistyowati, Sri; Hadi, Sudharto P.

    2018-02-01

    High Conservation Value Forest (HCVF) is the identification of High Conservation Values that are important and need to be protected. Under FSC certification mechanism, HCVF becomes one of Principles and Criteria to attain certification. In this study, we identify the existence of HCVF in Perum Perhutani KPH Kendal to support implementation process of FSC certification. Qualitative method was conducted through observation and secondary data from Perum Perhutani KPH Kendal. Data analysis showed through ecolabel certification, Perum Perhutani KPH Kendal has been identified HCVF area covering 2,715.5 hectares consists of HCV 1 until 6. Secondary Natural Forest (HAS) Subah and Kaliwungu for Ulolanang and Pagerwunung Nature Reserve buffer zone include as HCV 1.1, conservation area of leopard (Panthera pardus melas) and Pangolin (Manis javanica).for HCV 1.2, conservation area of lutung (Trachypiyhecus auratus) as endemic species for CITES App I and Critically Endangered species include as HCV 1.3, Goa kiskendo for bats species habitat include as HCV 1.4, regions of interest species for Deer (Cervus timorensis) and Kepodang (Oriolus chinensis) as HCV 2.3, Germplasm Protection Region/ KPPN area with high biodiversity include as HCV 3, river border area and water springs for HCV 4. While, utilization of firewood, grass for cattle fodder include as HCV 5 and 14 cultural sites include as HCV 6. From monitoring and evaluation of HCVF data, showed that in 2011-2015 the level of diversity for flora and fauna were increased.

  11. DEVELOPING OF INSTRUCTIONAL MEDIA-BASED ANIMATION VIDEO ON ENZYME AND METABOLISM MATERIAL IN SENIOR HIGH SCHOOL

    Directory of Open Access Journals (Sweden)

    Muhammad Mustofa Yusuf

    2017-11-01

    Full Text Available The research aimed to product a learning material related to animation video on enzyme and metabolism material for high school student which is validated by media and material experts, educational practition and student legibility. Research and development model is ADDIE with quantitative-qualitative data analyzing methode. Data collection was obtained from validation results by media and material experts, educational partition and student legibility. The validation results were scores and suggestion. The percentage of product from expert media validation (100%, expert material validation (89,58%, educational practition (84,61%, and student legibility (81,91% showed valid of the criteria and feasible to use after revision.

  12. Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Yanase, Shuhei; Yamada, Ryosuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Hasunuma, Tomohisa; Tanaka, Tsutomu; Fukuda, Hideki [Kobe Univ. (Japan). Organization of Advanced Science and Technology

    2010-09-15

    To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 C and 37 C, while the activity of cellulolytic enzymes is highest at around 50 C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus {beta}-glucosidase on the cell surface, which successfully converts a cellulosic {beta}-glucan to ethanol directly at 48 C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of {beta}-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface. (orig.)

  13. Development of high-performance functional materials for enzyme immobilization by the use of ionizing radiation

    International Nuclear Information System (INIS)

    SALIM, R.D.M.

    2013-01-01

    Isomerization of glucose to fructose was carried out using Glucose isomerase (GI) that immobilized by entrapment into Poly (acrylic acid) P (AA) and Poly (acrylic acid-co- 2-Acrylamido 2- methyl Propane sulfonic acid) P (AA-co-AMPS) polymer networks, the enzyme carriers were prepared by radiation induced co-polymerization in presence of (Methylene- bis acrylamide) (MBAA) as a crosslinking agent. Effects of immobilization conditions such as irradiation dose, methylene bis acrylamide concentration, comonomer composition, and amount of GI were investigated. The influence of reaction conditions on the activity of immobilized GI were studied, the optimum ph value of reaction solution is 7.5 and reaction temperature is 65 degree C. The immobilized GI into P (AA-co-AMPS) and P (AA) polymer networks retained 81% and 69%,respectively, of its initial activity after recycled for 15 times while it retained 87% and 71% ,respectively ,of its initial activity after stored at 4 degree C for 48 days , The Km values of free and immobilized GI onto P(AA-co-AMPS) and onto P(AA) matrices were found to be 34, 29.2 , 14.5 mg/ml respectively while the Vmax Values calculated to be 3.87 ,1.6,0.79 mg/ml.min, respectively, Therefore , the bio conversion of glucose to fructose can be successfully performed by GI entrapped into P (AA-co-AMPS) hydrogel .

  14. High activity antioxidant enzymes protect flying-fox haemoglobin against damage: an evolutionary adaptation for flight?

    Science.gov (United States)

    Reinke, N B; O'Brien, G M

    2006-11-01

    Flying-foxes are better able to defend haemoglobin against autoxidation than non-volant mammals such as sheep. When challenged with the common physiological oxidant, hydrogen peroxide, haemolysates of flying-fox red blood cells (RBC) were far less susceptible to methaemoglobin formation than sheep. Challenge with 1-acetyl-2-phenylhydrazine (APH) caused only half as much methaemoglobin formation in flying-fox as in ovine haemolysates. When intact cells were challenged with phenazine methosulfate (PMS), flying-fox RBC partially reversed the oxidant damage, and reduced methaemoglobin from 40 to 20% over 2 h incubation, while ovine methaemoglobin remained at 40%. This reflected flying-fox cells' capacity to replenish GSH fast enough that it did not deplete beyond 50%, while ovine RBC GSH was depleted to around 20%. The greater capacity of flying-foxes to defend haemoglobin against oxidant damage may be explained in part by antioxidant enzymes catalase, superoxide dismutase and cytochrome-b ( 5 ) reductase having two- to four-fold higher activity than in sheep (P foxes.

  15. High-frequency ultrasound for intraoperative margin assessments in breast conservation surgery: a feasibility study

    International Nuclear Information System (INIS)

    Doyle, Timothy E; Neumayer, Leigh A; Factor, Rachel E; Ellefson, Christina L; Sorensen, Kristina M; Ambrose, Brady J; Goodrich, Jeffrey B; Hart, Vern P; Jensen, Scott C; Patel, Hemang

    2011-01-01

    In addition to breast imaging, ultrasound offers the potential for characterizing and distinguishing between benign and malignant breast tissues due to their different microstructures and material properties. The aim of this study was to determine if high-frequency ultrasound (20-80 MHz) can provide pathology sensitive measurements for the ex vivo detection of cancer in margins during breast conservation surgery. Ultrasonic tests were performed on resected margins and other tissues obtained from 17 patients, resulting in 34 specimens that were classified into 15 pathology categories. Pulse-echo and through-transmission measurements were acquired from a total of 57 sites on the specimens using two single-element 50-MHz transducers. Ultrasonic attenuation and sound speed were obtained from time-domain waveforms. The waveforms were further processed with fast Fourier transforms to provide ultrasonic spectra and cepstra. The ultrasonic measurements and pathology types were analyzed for correlations. The specimens were additionally re-classified into five pathology types to determine specificity and sensitivity values. The density of peaks in the ultrasonic spectra, a measure of spectral structure, showed significantly higher values for carcinomas and precancerous pathologies such as atypical ductal hyperplasia than for normal tissue. The slopes of the cepstra for non-malignant pathologies displayed significantly greater values that differentiated them from the normal and malignant tissues. The attenuation coefficients were sensitive to fat necrosis, fibroadenoma, and invasive lobular carcinoma. Specificities and sensitivities for differentiating pathologies from normal tissue were 100% and 86% for lobular carcinomas, 100% and 74% for ductal carcinomas, 80% and 82% for benign pathologies, and 80% and 100% for fat necrosis and adenomas. Specificities and sensitivities were also determined for differentiating each pathology type from the other four using a multivariate

  16. High-frequency ultrasound for intraoperative margin assessments in breast conservation surgery: a feasibility study

    Directory of Open Access Journals (Sweden)

    Hart Vern P

    2011-10-01

    Full Text Available Abstract Background In addition to breast imaging, ultrasound offers the potential for characterizing and distinguishing between benign and malignant breast tissues due to their different microstructures and material properties. The aim of this study was to determine if high-frequency ultrasound (20-80 MHz can provide pathology sensitive measurements for the ex vivo detection of cancer in margins during breast conservation surgery. Methods Ultrasonic tests were performed on resected margins and other tissues obtained from 17 patients, resulting in 34 specimens that were classified into 15 pathology categories. Pulse-echo and through-transmission measurements were acquired from a total of 57 sites on the specimens using two single-element 50-MHz transducers. Ultrasonic attenuation and sound speed were obtained from time-domain waveforms. The waveforms were further processed with fast Fourier transforms to provide ultrasonic spectra and cepstra. The ultrasonic measurements and pathology types were analyzed for correlations. The specimens were additionally re-classified into five pathology types to determine specificity and sensitivity values. Results The density of peaks in the ultrasonic spectra, a measure of spectral structure, showed significantly higher values for carcinomas and precancerous pathologies such as atypical ductal hyperplasia than for normal tissue. The slopes of the cepstra for non-malignant pathologies displayed significantly greater values that differentiated them from the normal and malignant tissues. The attenuation coefficients were sensitive to fat necrosis, fibroadenoma, and invasive lobular carcinoma. Specificities and sensitivities for differentiating pathologies from normal tissue were 100% and 86% for lobular carcinomas, 100% and 74% for ductal carcinomas, 80% and 82% for benign pathologies, and 80% and 100% for fat necrosis and adenomas. Specificities and sensitivities were also determined for differentiating each

  17. Geometrid moth assemblages reflect high conservation value of naturally regenerated secondary forests in temperate China

    NARCIS (Netherlands)

    Zou, Yi; Sang, Weiguo; Warren-Thomas, Eleanor; Axmacher, Jan Christoph

    2016-01-01

    The widespread destruction of mature forests in China has led to massive ecological degradation, counteracted in recent decades by substantial efforts to promote forest plantations and protect secondary forest ecosystems. The value of the resulting forests for biodiversity conservation is widely

  18. Liberals, Conservatives and Romantic Nationalists in Interwar Education Policy in Greece: "The High Mountains" Episode

    Science.gov (United States)

    Athanasiades, Harris

    2015-01-01

    Greek historiography of interwar education policy unproblematically accepts the assumption that the bone of contention between the "Liberal demoticists" and the "Conservative purists" was the language issue; particularly whether "demotic" or "katharevousa" should be the language of instruction in schooling.…

  19. Gene co-regulation is highly conserved in the evolution of eukaryotes and prokaryotes.

    NARCIS (Netherlands)

    Snel, B.; Noort, V. van; Huynen, M.A.

    2004-01-01

    Differences between species have been suggested to largely reside in the network of connections among the genes. Nevertheless, the rate at which these connections evolve has not been properly quantified. Here, we measure the extent to which co-regulation between pairs of genes is conserved over

  20. GROWTH AND ENZYME PRODUCTION DURING CONTINUOUS CULTURES OF A HIGH AMYLASE-PRODUCING VARIANT OF Aspergillus Oryzae

    Directory of Open Access Journals (Sweden)

    T.C. Zangirolami

    2002-03-01

    Full Text Available Growth and product formation by a selected variant of Aspergillus oryzae showing high alpha-amylase production was studied in continuous cultivations carried out at six different specific growth rates, using glucose as the growth-limiting nutrient. The analysis of the steady-state data revealed that the variant and wild-type strains were similar with respect to glucose uptake system and stoichiometric coefficients. However, the variant was capable of maintaining an enzyme production as high as 40 FAUgDW-1h-1 at a dilution rate of 0.2 h-1, while the wild-type strain reached a maximum specific alpha-amylase production rate of 17 FAUgDW-1h-1 at a dilution rate of 0.1 h-1. Using a morphologically structured model originally proposed for the wild-type strain, it was possible to describe enzyme production, biomass formation and glucose consumption after modification of a few parameters to adjust the model to the characteristics of the selected variant.

  1. Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation.

    Science.gov (United States)

    Boshoff, Sonja; Gottumukkala, Lalitha Devi; van Rensburg, Eugéne; Görgens, Johann

    2016-03-01

    Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5 g/L at 78.2%, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Androgen receptor status is highly conserved during tumor progression of breast cancer.

    Science.gov (United States)

    Grogg, André; Trippel, Mafalda; Pfaltz, Katrin; Lädrach, Claudia; Droeser, Raoul A; Cihoric, Nikola; Salhia, Bodour; Zweifel, Martin; Tapia, Coya

    2015-11-09

    highly conserved during tumor progression and a change only occurs in a small fraction (4.1 %). Our study supports the notion that targeting AR could be effective for many BC patients and that re-testing of AR status in formerly negative or mixed type BC's is recommended.

  3. Androgen receptor status is highly conserved during tumor progression of breast cancer

    International Nuclear Information System (INIS)

    Grogg, André; Trippel, Mafalda; Pfaltz, Katrin; Lädrach, Claudia; Droeser, Raoul A.; Cihoric, Nikola; Salhia, Bodour; Zweifel, Martin; Tapia, Coya

    2015-01-01

    With the advent of new and more efficient anti-androgen drugs targeting androgen receptor (AR) in breast cancer (BC) is becoming an increasingly important area of investigation. This would potentially be most useful in triple negative BC (TNBC), where better therapies are still needed. The assessment of AR status is generally performed on the primary tumor even if the tumor has already metastasized. Very little is known regarding discrepancies of AR status during tumor progression. To determine the prevalence of AR positivity, with emphasis on TNBCs, and to investigate AR status during tumor progression, we evaluated a large series of primary BCs and matching metastases and recurrences. AR status was performed on 356 primary BCs, 135 matching metastases, and 12 recurrences using a next-generation Tissue Microarray (ngTMA). A commercially available AR antibody was used to determine AR-status by immunohistochemistry. AR positivity was defined as any nuclear staining in tumor cells ≥1 %. AR expression was correlated with pathological tumor features of the primary tumor. Additionally, the concordance rate of AR expression between the different tumor sites was determined. AR status was positive in: 87 % (307/353) of primary tumors, 86.1 % (105/122) of metastases, and in 66.7 % (8/12) of recurrences. TNBC tested positive in 11.4 %, (4/35) of BCs. A discrepant result was seen in 4.3 % (5/117) of primary BC and matching lymph node (LN) metastases. Three AR negative primary BCs were positive in the matching LN metastasis, representing 17.6 % of all negative BCs with lymph node metastases (3/17). Two AR positive primary BCs were negative in the matching LN metastasis, representing 2.0 % of all AR positive BCs with LN metastases (2/100). No discrepancies were seen between primary BC and distant metastases or recurrence (n = 17). Most primary (87 %) and metastasized (86.1 %) BCs are AR positive including a significant fraction of TNBCs (11.4 %). Further, AR status is highly

  4. Conservation hotspots for the turtles on the high seas of the Atlantic Ocean.

    Directory of Open Access Journals (Sweden)

    Hsiang-Wen Huang

    Full Text Available Understanding the distribution of bycaught sea turtles could inform conservation strategies and priorities. This research analyses the distribution of turtles caught as longline fisheries bycatch on the high seas of the Atlantic Ocean. This research collected 18,142 bycatch observations and 47.1 million hooks from large-scale Taiwanese longline vessels in the Atlantic Ocean from June 2002 to December 2013. The coverage rates were ranged from 0.48% to 17.54% by year. Seven hundred and sixty-seven turtles were caught, and the major species were leatherback (59.8%, olive ridley (27.1% and loggerhead turtles (8.7%. Most olive ridley (81.7% and loggerhead (82.1% turtles were hooked, while the leatherbacks were both hooked (44.0% and entangled (31.8%. Depending on the species, 21.4% to 57.7% were dead when brought onboard. Most of the turtles were caught in tropical areas, especially in the Gulf of Guinea (15°N-10°S, 30°W-10°E, but loggerheads were caught in the south Atlantic Ocean (25°S-35°S, 40°W-10°E and 30°S-40°S, 55°W-45°W. The bycatch rate was the highest at 0.030 per 1000 hooks for leatherbacks in the tropical area. The bycatch rates of olive ridley ranged from 0 to 0.010 per thousand hooks. The loggerhead bycatch rates were higher in the northern and southern Atlantic Ocean and ranged from 0.0128 to 0.0239 per thousand hooks. Due to the characteristics of the Taiwanese deep-set longline fleet, bycatch rates were lower than those of coastal longline fisheries, but mortality rates were higher because of the long hours of operation. Gear and bait modification should be considered to reduce sea turtle bycatch and increase survival rates while reducing the use of shallow hooks would also be helpful.

  5. Conservation hotspots for the turtles on the high seas of the Atlantic Ocean.

    Science.gov (United States)

    Huang, Hsiang-Wen

    2015-01-01

    Understanding the distribution of bycaught sea turtles could inform conservation strategies and priorities. This research analyses the distribution of turtles caught as longline fisheries bycatch on the high seas of the Atlantic Ocean. This research collected 18,142 bycatch observations and 47.1 million hooks from large-scale Taiwanese longline vessels in the Atlantic Ocean from June 2002 to December 2013. The coverage rates were ranged from 0.48% to 17.54% by year. Seven hundred and sixty-seven turtles were caught, and the major species were leatherback (59.8%), olive ridley (27.1%) and loggerhead turtles (8.7%). Most olive ridley (81.7%) and loggerhead (82.1%) turtles were hooked, while the leatherbacks were both hooked (44.0%) and entangled (31.8%). Depending on the species, 21.4% to 57.7% were dead when brought onboard. Most of the turtles were caught in tropical areas, especially in the Gulf of Guinea (15°N-10°S, 30°W-10°E), but loggerheads were caught in the south Atlantic Ocean (25°S-35°S, 40°W-10°E and 30°S-40°S, 55°W-45°W). The bycatch rate was the highest at 0.030 per 1000 hooks for leatherbacks in the tropical area. The bycatch rates of olive ridley ranged from 0 to 0.010 per thousand hooks. The loggerhead bycatch rates were higher in the northern and southern Atlantic Ocean and ranged from 0.0128 to 0.0239 per thousand hooks. Due to the characteristics of the Taiwanese deep-set longline fleet, bycatch rates were lower than those of coastal longline fisheries, but mortality rates were higher because of the long hours of operation. Gear and bait modification should be considered to reduce sea turtle bycatch and increase survival rates while reducing the use of shallow hooks would also be helpful.

  6. A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

    DEFF Research Database (Denmark)

    Kracun, Stjepan Kresimir; Schückel, Julia; Westereng, Bjørge

    2015-01-01

    of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results: We have developed a new generation of multi...

  7. High Resolution Mapping of Soils and Landforms for the Desert Renewable Energy Conservation Plan (DRECP)

    Science.gov (United States)

    Potter, Christopher S.; Li, Shuang

    2014-01-01

    The Desert Renewable Energy Conservation Plan (DRECP), a major component of California's renewable energy planning efforts, is intended to provide effective protection and conservation of desert ecosystems, while allowing for the sensible development of renewable energy projects. This NASA mapping report was developed to support the DRECP and the Bureau of Land Management (BLM). We outline in this document remote sensing image processing methods to deliver new maps of biological soils crusts, sand dune movements, desert pavements, and sub-surface water sources across the DRECP area. We focused data processing first on the largely unmapped areas most likely to be used for energy developments, such as those within Renewable Energy Study Areas (RESA) and Solar Energy Zones (SEZs). We used imagery (multispectral and radar) mainly from the years 2009-2011.

  8. High-Quality 3d Models and Their Use in a Cultural Heritage Conservation Project

    Science.gov (United States)

    Tucci, G.; Bonora, V.; Conti, A.; Fiorini, L.

    2017-08-01

    Cultural heritage digitization and 3D modelling processes are mainly based on laser scanning and digital photogrammetry techniques to produce complete, detailed and photorealistic three-dimensional surveys: geometric as well as chromatic aspects, in turn testimony of materials, work techniques, state of preservation, etc., are documented using digitization processes. The paper explores the topic of 3D documentation for conservation purposes; it analyses how geomatics contributes in different steps of a restoration process and it presents an overview of different uses of 3D models for the conservation and enhancement of the cultural heritage. The paper reports on the project to digitize the earthenware frieze of the Ospedale del Ceppo in Pistoia (Italy) for 3D documentation, restoration work support, and digital and physical reconstruction and integration purposes. The intent to design an exhibition area suggests new ways to take advantage of 3D data originally acquired for documentation and scientific purposes.

  9. A High-Accuracy Linear Conservative Difference Scheme for Rosenau-RLW Equation

    Directory of Open Access Journals (Sweden)

    Jinsong Hu

    2013-01-01

    Full Text Available We study the initial-boundary value problem for Rosenau-RLW equation. We propose a three-level linear finite difference scheme, which has the theoretical accuracy of Oτ2+h4. The scheme simulates two conservative properties of original problem well. The existence, uniqueness of difference solution, and a priori estimates in infinite norm are obtained. Furthermore, we analyze the convergence and stability of the scheme by energy method. At last, numerical experiments demonstrate the theoretical results.

  10. Hearing sensitivity in context: Conservation implications for a highly vocal endangered species

    OpenAIRE

    Owen, Megan A.; Keating, Jennifer L.; Denes, Samuel K.; Hawk, Kathy; Fiore, Angela; Thatcher, Julie; Becerra, Jennifer; Hall, Suzanne; Swaisgood, Ronald R.

    2016-01-01

    Hearing sensitivity is a fundamental determinant of a species’ vulnerability to anthropogenic noise, however little is known about the hearing capacities of most conservation dependent species. When audiometric data are integrated with other aspects of species’ acoustic ecology, life history, and characteristic habitat topography and soundscape, predictions can be made regarding probable vulnerability to the negative impacts of different types of anthropogenic noise. Here we used an adaptive ...

  11. Innovative Surgical Management of the Synovial Chondromatosis of Temporo-Mandibular Joints: Highly Conservative Surgical Technique.

    Science.gov (United States)

    Ionna, Franco; Amantea, Massimiliano; Mastrangelo, Filiberto; Ballini, Andrea; Maglione, Maria Grazia; Aversa, Corrado; De Cecio, Rossella; Russo, Daniela; Marrelli, Massimo; Tatullo, Marco

    2016-07-01

    Synovial chondromatosis (SC) is an uncommon disease characterized by a benign nodular cartilaginous proliferation arising from the joint synovium, bursae, or tendon sheaths. Although the temporomandibular joint is rarely affected by neoplastic lesions, SC is the most common neoplastic lesion of this joint. The treatment of this disease consists in the extraoral surgery with a wide removal of the lesion; in this study, the authors described a more conservative intraoral surgical approach. Patient with SC of temporomandibular joint typically refer a limitation in the mouth opening, together with a persistent not physiological mandibular protrusion and an appearance of a neoformation located at the right preauricular region: the authors reported 1 scholar patient. After biopsy of the neoformation, confirming the synovial chondromatosis, the patient underwent thus to the surgical excision of the tumor, via authors' conservative transoral approach, to facilitate the enucleation of the neoformation. The mass fully involved the pterygo-maxillary fossa with involvement of the parotid lodge and of the right TMJ: this multifocal extension suggested for a trans-oral surgical procedure, in the light of the suspicion of a possible malignant nature of the neoplasm. Our intraoral conservative approach to surgery is aimed to reduce the presence of unaesthetic scars in preauricular and facial regions, with surgical results undoubtedly comparable to the traditional surgical techniques much more aggressive. Our technique could be a valid, alternative, and safe approach to treat this rare and complex kind of oncological disease.

  12. Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.

    Science.gov (United States)

    Sun, Shuwen; Kennedy, Robert T

    2014-09-16

    High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a "MS Plate Reader" that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high Z-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145,000 samples to be analyzed in 1 day.

  13. High performance separation of xylose and glucose by enzyme assisted nanofiltration

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Luo, Jianquan; Meyer, Anne S.

    2015-01-01

    of the integrated system. Full conversion of glucose to gluconic acid assisted by glucose oxidase (GOD) could be achieved by coupling a parallel reaction catalyzed by catalase (CAT), where H2O2 (GOD-inhibitor formed in the first reaction) was decomposed to water and oxygen. GOD has a high oxygen...

  14. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  15. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  16. NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

    Science.gov (United States)

    de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

    2013-01-01

    NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

  17. The importance of incorporating functional habitats into conservation planning for highly mobile species in dynamic systems.

    Science.gov (United States)

    Webb, Matthew H; Terauds, Aleks; Tulloch, Ayesha; Bell, Phil; Stojanovic, Dejan; Heinsohn, Robert

    2017-10-01

    The distribution of mobile species in dynamic systems can vary greatly over time and space. Estimating their population size and geographic range can be problematic and affect the accuracy of conservation assessments. Scarce data on mobile species and the resources they need can also limit the type of analytical approaches available to derive such estimates. We quantified change in availability and use of key ecological resources required for breeding for a critically endangered nomadic habitat specialist, the Swift Parrot (Lathamus discolor). We compared estimates of occupied habitat derived from dynamic presence-background (i.e., presence-only data) climatic models with estimates derived from dynamic occupancy models that included a direct measure of food availability. We then compared estimates that incorporate fine-resolution spatial data on the availability of key ecological resources (i.e., functional habitats) with more common approaches that focus on broader climatic suitability or vegetation cover (due to the absence of fine-resolution data). The occupancy models produced significantly (P increase or decrease in the area of one functional habitat (foraging or nesting) did not necessarily correspond to an increase or decrease in the other. Thus, an increase in the extent of occupied area may not equate to improved habitat quality or function. We argue these patterns are typical for mobile resource specialists but often go unnoticed because of limited data over relevant spatial and temporal scales and lack of spatial data on the availability of key resources. Understanding changes in the relative availability of functional habitats is crucial to informing conservation planning and accurately assessing extinction risk for mobile resource specialists. © 2017 Society for Conservation Biology.

  18. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

    Directory of Open Access Journals (Sweden)

    Quevillon-Cheruel Sophie

    2007-01-01

    Full Text Available Abstract The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes.

  19. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    Science.gov (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  20. High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

    DEFF Research Database (Denmark)

    Kang, Aram; Meadows, Corey W.; Canu, Nicolas

    2017-01-01

    Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high...... ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass� isopentenol pathway using the promiscuous activity...

  1. Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

    Directory of Open Access Journals (Sweden)

    Ouyang Shu

    2005-09-01

    Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

  2. Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

    Science.gov (United States)

    Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

    2017-04-01

    This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm 2 of one banknote by swabbing 1cm 2 : 0, 14, and 21pg/cm 2 . Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

    Directory of Open Access Journals (Sweden)

    Shahin Ranjbar

    2006-12-01

    Full Text Available To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5, which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.

  4. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  5. The conservation value of South East Asia's highly degraded forests: evidence from leaf-litter ants

    Science.gov (United States)

    Woodcock, Paul; Edwards, David P.; Fayle, Tom M.; Newton, Rob J.; Khen, Chey Vun; Bottrell, Simon H.; Hamer, Keith C.

    2011-01-01

    South East Asia is widely regarded as a centre of threatened biodiversity owing to extensive logging and forest conversion to agriculture. In particular, forests degraded by repeated rounds of intensive logging are viewed as having little conservation value and are afforded meagre protection from conversion to oil palm. Here, we determine the biological value of such heavily degraded forests by comparing leaf-litter ant communities in unlogged (natural) and twice-logged forests in Sabah, Borneo. We accounted for impacts of logging on habitat heterogeneity by comparing species richness and composition at four nested spatial scales, and examining how species richness was partitioned across the landscape in each habitat. We found that twice-logged forest had fewer species occurrences, lower species richness at small spatial scales and altered species composition compared with natural forests. However, over 80 per cent of species found in unlogged forest were detected within twice-logged forest. Moreover, greater species turnover among sites in twice-logged forest resulted in identical species richness between habitats at the largest spatial scale. While two intensive logging cycles have negative impacts on ant communities, these degraded forests clearly provide important habitat for numerous species and preventing their conversion to oil palm and other crops should be a conservation priority. PMID:22006966

  6. Application of high hydrostatic pressure for increasing activity and stability of enzymes.

    Science.gov (United States)

    Mozhaev, V V; Lange, R; Kudryashova, E V; Balny, C

    1996-10-20

    Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the

  7. Classification of lipolytic enzymes and their biotechnological applications in the pulping industry

    CSIR Research Space (South Africa)

    Ramnath, L

    2017-03-01

    Full Text Available are very closely related (Lee 2016). Enzymes exhibit the canon- ical�/�-hydrolase fold and contain a typical catalytic triad. High activities at low temperature (less than 15 °C) were believed to originate from conserved sequence motifs of these enzymes... enzymes to a family. However, unique families are being discovered through the use of metagenomics (Fu et al. 2011; Kim et al. 2009; Lee et al. 2006). Table 1 summarizes the different classes of lipo- lytic enzymes currently described. Lipases Lipases (e...

  8. 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 hydrolases...... that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes...

  9. Growth responses of native chicken Sentul G-3 on diet containing high rice-bran supplemented with phytase enzyme and ZnO

    Directory of Open Access Journals (Sweden)

    Cecep Hidayat

    2014-10-01

    Full Text Available This study was conducted to determine the effect of phytase enzymes and ZnO supplementation on the performance of native chicken Sentul-G3 fed high rice-bran diet. Two hundred and seventy day old chicks (DOC native chicken Sentul-G3 from three different hatcheries were used in this study. Factorial randomized block design (3 x 3 was applied in this study. The first factor was the enzyme phytase supplementation levels (0; 1000; 2000 U/kg, the second factor was the level of supplementation of ZnO (0; 1.5; 3.2 g/kg, so that there are nine treatment given, namely R1 = 50% commercial diet : 50% rice bran; R2 = R1 + 1.5 g ZnO/kg; R3 = R1 + 3.2 g ZnO/kg; R4 = R1 + phytase enzyme 1000 U/kg; R5 = R1 + (phytase enzyme 1000 U/kg + 1.5 g ZnO/kg; R6 = R1 + (phytase enzyme 1000 U/kg + 3.2 g ZnO/kg; R7 = R1 + phytase enzyme 2000 U/kg; R8 = R1 + (phytase enzyme 2000 U/kg + 1.5 g ZnO/kg; R9 = R1 + (phytase enzyme 2000 U/kg + 3.2 g ZnO/kg. Each experimental unit consisted of 6 head unsexed native chicken Sentul-G3. The experimental diet was fed for 10 weeks. The variables measured were body weight, body weight gain, feed intake, feed conversion ratio, mortality, mineral deposition of Ca, P, Zn in the tibia bone, alkaline phosfatase enzyme activity in serum. Results showed that there was significant interaction (P 0.05 between phytase enzyme and ZnO supplementation on feed intake, mortality, alkaline phosphatase enzyme activity in serum, and deposition of calcium and phosphorus in the tibia bone. It was concluded that supplementation of phytase enzyme and ZnO were not able to increase the growth of native chicken Sentul-G3 on fed diet containing high rice bran.

  10. Impacts of Tropical Forest Disturbance Upon Avifauna on a Small Island with High Endemism: Implications for Conservation

    Directory of Open Access Journals (Sweden)

    Martin Thomas

    2010-01-01

    Full Text Available Tropical forests are rapidly being lost across Southeast Asia and this is predicted to have severe implications for many of the region′s bird species. However, relationships between forest disturbance and avifaunal assemblages remain poorly understood, particularly on small island ecosystems such as those found in the biodiversity ′hotspot′ of Wallacea. This study examines how avifaunal richness varies across a disturbance gradient in a forest reserve on Buton Island, southeast Sulawesi. Particular emphasis is placed upon examining responses in endemic and red-listed species with high conservation importance. Results indicate that overall avian richness increases between primary and 30-year-old regenerating secondary forest and then decreases through disturbed secondary forest, but is highest in cleared farmland. However, high species richness in farmland does not signify high species distinctiveness; bird community composition here differs significantly from that found in forest sites, and is poor in supporting forest specialists and endemic species. Certain large-bodied endemics such as the Knobbed Hornbill (Rhyticeros cassidix appear to be sensitive to moderate disturbance, with populations occurring at greatest density within primary forest. However, overall endemic species richness, as well as that of endemic frugivores and insectivores, is similar in primary and secondary forest types. Results indicate that well-established secondary forest in particular has an important role in supporting species with high conservational importance, possessing community composition similar to that found in primary forest and supporting an equally high richness of endemic species.

  11. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    International Nuclear Information System (INIS)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung; Koo, Bonsung

    2013-01-01

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS

  12. Noise exposure and hearing conservation practices in an industry with high incidence of workers' compensation claims for hearing loss.

    Science.gov (United States)

    Daniell, William E; Swan, Susan S; McDaniel, Mary M; Stebbins, John G; Seixas, Noah S; Morgan, Michael S

    2002-10-01

    Washington State has experienced a striking increase in workers' compensation claims for hearing loss. This cross-sectional study examined noise exposures and hearing conservation practices in one industry with a high rate of hearing loss claims. We evaluated 10 representative foundries with personal noise dosimetry, management interviews, employee interviews, and existing audiometry. Noise levels routinely exceeded 85 dBA. All companies were out of compliance with hearing conservation regulations. Most employees with important findings on audiograms were not aware of their findings. There was a significant positive correlation between management-interview scores and worksite-average employee-interview scores (r = 0.70, P = 0.02). Companies where more effort is put into hearing conservation program activities can achieve a greater positive impact on employee awareness. However, there were broad deficiencies even in the better programs in this sample, suggesting that workers in this industry probably face a continuing substantial risk of occupational hearing loss. Copyright 2002 Wiley-Liss, Inc.

  13. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    Energy Technology Data Exchange (ETDEWEB)

    Hashem, Anwar M. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah (Saudi Arabia); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada); Van Domselaar, Gary [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Li, Changgui; Wang, Junzhi [National Institute for the Control of Pharmaceutical and Biological Products, Beijing (China); She, Yi-Min; Cyr, Terry D. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Sui, Jianhua [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); He, Runtao [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Marasco, Wayne A. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Li, Xuguang, E-mail: Sean.Li@hc-sc.gc.ca [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada)

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  14. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    International Nuclear Information System (INIS)

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-01-01

    Research highlights: → The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. → Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. → The universal antibodies cross-neutralize different influenza A subtypes. → The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  15. A high-order relaxation method with projective integration for solving nonlinear systems of hyperbolic conservation laws

    Science.gov (United States)

    Lafitte, Pauline; Melis, Ward; Samaey, Giovanni

    2017-07-01

    We present a general, high-order, fully explicit relaxation scheme which can be applied to any system of nonlinear hyperbolic conservation laws in multiple dimensions. The scheme consists of two steps. In a first (relaxation) step, the nonlinear hyperbolic conservation law is approximated by a kinetic equation with stiff BGK source term. Then, this kinetic equation is integrated in time using a projective integration method. After taking a few small (inner) steps with a simple, explicit method (such as direct forward Euler) to damp out the stiff components of the solution, the time derivative is estimated and used in an (outer) Runge-Kutta method of arbitrary order. We show that, with an appropriate choice of inner step size, the time step restriction on the outer time step is similar to the CFL condition for the hyperbolic conservation law. Moreover, the number of inner time steps is also independent of the stiffness of the BGK source term. We discuss stability and consistency, and illustrate with numerical results (linear advection, Burgers' equation and the shallow water and Euler equations) in one and two spatial dimensions.

  16. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  17. Anti-Inflammatory and Antioxidant Properties of Casein Hydrolysate Produced Using High Hydrostatic Pressure Combined with Proteolytic Enzymes.

    Science.gov (United States)

    Bamdad, Fatemeh; Shin, Seulki Hazel; Suh, Joo-Won; Nimalaratne, Chamila; Sunwoo, Hoon

    2017-04-10

    Casein-derived peptides are shown to possess radical scavenging and metal chelating properties. The objective of this study was to evaluate novel anti-inflammatory properties of casein hydrolysates (CH) produced by an eco-friendly process that combines high hydrostatic pressure with enzymatic hydrolysis (HHP-EH). Casein was hydrolysed by different proteases, including flavourzyme (Fla), savinase (Sav), thermolysin (Ther), trypsin (Try), and elastase (Ela) at 0.1, 50, 100, and 200 MPa pressure levels under various enzyme-to-substrate ratios and incubation times. Casein hydrolysates were evaluated for the degree of hydrolysis (DH), molecular weight distribution patterns, and anti-inflammatory properties in chemical and cellular models. Hydrolysates produced using HHP-EH exhibited higher DH values and proportions of smaller peptides compared to atmospheric pressure-enzymatic hydrolysis (AP-EH). Among five enzymes, Fla-digested HHP-EH-CH (HHP-Fla-CH) showed significantly higher antioxidant properties than AP-Fla-CH. The anti-inflammatory properties of HHP-Fla-CH were also observed by significantly reduced nitric oxide and by the suppression of the synthesis of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that 59% of the amino acids of the peptides in HHP-Fla-CH were composed of proline, valine, and leucine, indicating the potential anti-inflammatory properties. In conclusion, the HHP-EH method provides a promising technology to produce bioactive peptides from casein in an eco-friendly process.

  18. A new world monkey microsatellite (ap74) highly conserved in primates

    International Nuclear Information System (INIS)

    Oklander, Luciana Ines; Steinberg, Eliana Ruth; Dolores Mudry, Marta

    2012-01-01

    Given their great variability, microsatellites or STRS became the most commonly used genetic markers over the last 15 years. The analysis of these markers requires minimum quantities of DNA, allowing the use of noninvasive samples, such as feces or hair. We amplified the microsatellite ap74 in blood and hair samples in order to analyze the levels of genomic conservation among a wide range of primates including: lemur catta, alouatta caraya, ateles belzebuth, ateles chamek, pan troglodytes, papio sp., and Homo sapiens. in all cases we obtained amplification products that exhibited similar size both in monkeys and human (oscillating between 126 and 176 bp), except in the lemur where the detected fragment presented a size of approximately 1000 bp. the analysis of the nucleotide sequences permitted the evaluation of the molecular modifications experienced during the evolutionary process in primates.

  19. Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp.

    Science.gov (United States)

    Kaushik, Neelima; Nadella, Tejaswi; Rao, P Srinivasa

    2015-11-01

    This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6- to 20-min pressure-hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain. Exploration of additional hurdles such as pH, TSS, and temperature for enzyme inactivation during high pressure processing of fruits is useful from industrial point of view, as these parameters play key role in preservation process design. © 2015 Institute of Food Technologists®

  20. Some AFLP amplicons are highly conserved DNA sequences mapping to the same linkage groups in two F2 populations of carrot

    Directory of Open Access Journals (Sweden)

    Santos Carlos A.F.

    2002-01-01

    Full Text Available Amplified fragment length polymorphism (AFLP is a fast and reliable tool to generate a large number of DNA markers. In two unrelated F2 populations of carrot (Daucus carota L., Brasilia x HCM and B493 x QAL (wild carrot, it was hypothesized that DNA 1 digested with the same restriction endonuclease enzymes and amplified with the same primer combination and 2 sharing the same position in polyacrylamide gels should be conserved sequences. To test this hypothesis AFLP fragments from polyacrylamide gels were eluted, reamplified, separated in agarose gels, purified, cloned and sequenced. Among thirty-one paired fragments from each F2 population, twenty-six had identity greater than 91% and five presented identity of 24% to 44%. Among the twenty-six conserved AFLPs only one mapped to different linkage groups in the two populations while four of the five less-conserved bands mapped to different linkage groups. Of eight SCAR (sequence characterized amplified regions primers tested, one conserved AFLP resulted in co-dominant markers in both populations. Screening among 14 carrot inbreds or cultivars with three AFLP-SCAR primers revealed clear and polymorphic PCR products, with similar molecular sizes on agarose gels. The development of co-dominant markers based on conserved AFLP fragments will be useful to detect seed mixtures among hybrids, to improve and to merge linkage maps and to study diversity and phylogenetic relationships.

  1. Conservation and Role of Electrostatics in Thymidylate Synthase.

    Science.gov (United States)

    Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C

    2015-11-27

    Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

  2. Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay.

    Science.gov (United States)

    Pandit, Subrata; Kanjilal, Satyajyoti; Awasthi, Anshumali; Chaudhary, Anika; Banerjee, Dipankar; Bhatt, B N; Narwaria, Avinash; Singh, Rahul; Dutta, Kakoli; Jaggi, Manu; Singh, Anu T; Sharma, Neena; Katiyar, Chandra Kant

    2017-02-02

    Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress. Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay. Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes. Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (Pdrugs showed significantly (P<0.001and P<0.01) less or negligible HDI. Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated

  3. Hearing sensitivity in context: Conservation implications for a highly vocal endangered species

    Directory of Open Access Journals (Sweden)

    Megan A. Owen

    2016-04-01

    Full Text Available Hearing sensitivity is a fundamental determinant of a species’ vulnerability to anthropogenic noise, however little is known about the hearing capacities of most conservation dependent species. When audiometric data are integrated with other aspects of species’ acoustic ecology, life history, and characteristic habitat topography and soundscape, predictions can be made regarding probable vulnerability to the negative impacts of different types of anthropogenic noise. Here we used an adaptive psychoacoustic technique to measure hearing thresholds in the endangered giant panda; a species that uses acoustic communication to coordinate reproduction. Our results suggest that giant pandas have functional hearing into the ultrasonic range, with good sensitivity between 10.0 and 16.0 kHz, and best sensitivity measured at 12.5–14.0 kHz. We estimated the lower and upper limits of functional hearing as 0.10 and 70.0 kHz respectively. While these results suggest that panda hearing is similar to that of some other terrestrial carnivores, panda hearing thresholds above 14.0 kHz were significantly lower (i.e., more sensitive than those of the polar bear, the only other bear species for which data are available. We discuss the implications of this divergence, as well as the relationship between hearing sensitivity and the spectral parameters of panda vocalizations. We suggest that these data, placed in context, can be used towards the development of a sensory-based model of noise disturbance for the species.

  4. 77 FR 11785 - Energy Conservation Program: Public Meeting and Availability of the Framework Document for High...

    Science.gov (United States)

    2012-02-28

    ... standards for high-intensity discharge (HID) lamps. Accordingly, DOE will hold a public meeting to discuss..._standards/commercial/high_intensity_discharge_lamps.html . DATES: The Department will hold a public meeting... Technologies Program, Mailstop EE-2J, Framework Document for High-Intensity Discharge Lamps, EERE-2010-BT-STD...

  5. Surgical versus conservative treatment for high-risk stress fractures of the lower leg (anterior tibial cortex, navicular and fifth metatarsal base): a systematic review

    NARCIS (Netherlands)

    Mallee, Wouter H.; Weel, Hanneke; van Dijk, C. Niek; van Tulder, Maurits W.; Kerkhoffs, Gino M.; Lin, Chung-Wei Christine

    2015-01-01

    To compare surgical and conservative treatment for high-risk stress fractures of the anterior tibial cortex, navicular and proximal fifth metatarsal. Systematic searches of CENTRAL, MEDLINE, EMBASE, CINAHL, SPORTDiscus and PEDro were performed to identify relevant prospective and retrospective

  6. Alternate-Day High-Fat Diet Induces an Increase in Mitochondrial Enzyme Activities and Protein Content in Rat Skeletal Muscle.

    Science.gov (United States)

    Li, Xi; Higashida, Kazuhiko; Kawamura, Takuji; Higuchi, Mitsuru

    2016-04-06

    Long-term high-fat diet increases muscle mitochondrial enzyme activity and endurance performance. However, excessive calorie intake causes intra-abdominal fat accumulation and metabolic syndrome. The purpose of this study was to investigate the effect of an alternating day high-fat diet on muscle mitochondrial enzyme activities, protein content, and intra-abdominal fat mass in rats. Male Wistar rats were given a standard chow diet (CON), high-fat diet (HFD), or alternate-day high-fat diet (ALT) for 4 weeks. Rats in the ALT group were fed a high-fat diet and standard chow every other day for 4 weeks. After the dietary intervention, mitochondrial enzyme activities and protein content in skeletal muscle were measured. Although body weight did not differ among groups, the epididymal fat mass in the HFD group was higher than those of the CON and ALT groups. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities in the plantaris muscle of rats in HFD and ALT were significantly higher than that in CON rats, whereas there was no difference between HFD and ALT groups. No significant difference was observed in muscle glycogen concentration or glucose transporter-4 protein content among the three groups. These results suggest that an alternate-day high-fat diet induces increases in mitochondrial enzyme activities and protein content in rat skeletal muscle without intra-abdominal fat accumulation.

  7. Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation

    Science.gov (United States)

    Römling, Ute; Bian, Zhao; Hammar, Mårten; Sierralta, Walter D.; Normark, Staffan

    1998-01-01

    Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C) and csgDEFG, from S. typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli. The divergence of the curli region between S. typhimurium and E. coli at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently, S. typhimurium genes on low-copy-number plasmids were able to complement respective E. coli mutants, although not always to wild-type levels. rpoS and ompR are required for transcriptional activation of (at least) the csgD promoter. The high degree of conservation at the protein level and the identical regulation patterns in E. coli and S. typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species. PMID:9457880

  8. Conserved host response to highly pathogenic avian influenza virus infection in human cell culture, mouse and macaque model systems

    Directory of Open Access Journals (Sweden)

    McDermott Jason E

    2011-11-01

    Full Text Available Abstract Background Understanding host response to influenza virus infection will facilitate development of better diagnoses and therapeutic interventions. Several different experimental models have been used as a proxy for human infection, including cell cultures derived from human cells, mice, and non-human primates. Each of these systems has been studied extensively in isolation, but little effort has been directed toward systematically characterizing the conservation of host response on a global level beyond known immune signaling cascades. Results In the present study, we employed a multivariate modeling approach to characterize and compare the transcriptional regulatory networks between these three model systems after infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Using this approach we identified functions and pathways that display similar behavior and/or regulation including the well-studied impact on the interferon response and the inflammasome. Our results also suggest a primary response role for airway epithelial cells in initiating hypercytokinemia, which is thought to contribute to the pathogenesis of H5N1 viruses. We further demonstrate that we can use a transcriptional regulatory model from the human cell culture data to make highly accurate predictions about the behavior of important components of the innate immune system in tissues from whole organisms. Conclusions This is the first demonstration of a global regulatory network modeling conserved host response between in vitro and in vivo models.

  9. Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

    Science.gov (United States)

    Deguchi, Matsuo; Kagita, Masanori; Yoshioka, Nori; Tsukamoto, Hiroko; Takao, Miyuki; Tahara, Kazuko; Maeda, Ikuhiro; Hidaka, Yoh; Yamauchi, Satoshi; Kaneko, Atsushi; Miyakoshi, Hideo; Isomura, Mitsuo

    2017-10-06

    Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients. © 2017 Wiley Periodicals, Inc.

  10. Typing Candida albicans oral isolates from healthy Brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa

    Directory of Open Access Journals (Sweden)

    Marcelo Fabiano Gomes Boriollo

    2011-09-01

    Full Text Available The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis - MLEE and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival of strains. Two highly polymorphic and distantly genetically related taxa (A and B were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2 and clusters of moderately related strains (from I to X, suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation.

  11. Typing Candida albicans oral isolates from healthy brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa

    Science.gov (United States)

    Boriollo, Marcelo Fabiano Gomes; Spolidorio, Denise Madalena Palomari; Barros, Letizia Monteiro; Bassi, Rodrigo Carlos; Garcia, José Antonio Dias; Costa, Ana Maria Duarte Dias; Rosa, Edvaldo Antonio Ribeiro; Höfling, José Francisco

    2011-01-01

    The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis – MLEE) and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test) was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival) of strains. Two highly polymorphic and distantly genetically related taxa (A and B) were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2) and clusters of moderately related strains (from I to X), suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related) reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation. PMID:24031720

  12. Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

    Science.gov (United States)

    Aoyama, Akihisa; Kurane, Ryuichiro; Matsuura, Akira; Nagai, Kazuo

    2015-01-01

    An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

  13. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  14. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  15. High expression of ubiquitin-conjugating enzyme 2C (UBE2C) correlates with nasopharyngeal carcinoma progression

    International Nuclear Information System (INIS)

    Shen, Zhihua; Guo, Junli; Jie, Wei; Jiang, Xiaofan; Zeng, Chao; Zheng, Shaojiang; Luo, Botao; Zeng, Yumei; Ding, Ranran; Jiang, Hanguo; He, Qiyi

    2013-01-01

    Overexpression of ubiquitin-conjugating enzyme 2C (UBE2C) has been detected in many types of human cancers, and is correlated with tumor malignancy. However, the role of UBE2C in human nasopharyngeal carcinoma (NPC) is unclear. In this study, we investigated the role of aberrant UBE2C expression in the progression of human NPC. Immunohistochemical analysis was performed to detect UBE2C protein in clinical samples of NPC and benign nasopharyngeal tissues, and the association of UBE2C expression with patient clinicopathological characteristics was analyzed. UBEC2 expression profiles were evaluated in cell lines representing varying differentiated stages of NPC and immortalized nasopharyngeal epithelia NP-69 cells using quantitative RT-PCR, western blotting and fluorescent staining. Furthermore, UBE2C was knocked down using RNA interference in these cell lines and proliferation and cell cycle distribution was investigated. Immunohistochemical analysis revealed that UBE2C protein expression levels were higher in NPC tissues than in benign nasopharyngeal tissues (P<0.001). Moreover, high UBE2C protein expression was positively correlated with tumor size (P=0.017), lymph node metastasis (P=0.016) and distant metastasis (P=0.015) in NPC patients. In vitro experiments demonstrated that UBE2C expression levels were inversely correlated with the degree of differentiation of NPC cell lines, whereas UBE2C displayed low level of expression in NP-69 cells. Knockdown of UBE2C led to significant arrest at the S and G2/M phases of the cell cycle, and decreased cell proliferation was observed in poorly-differentiated CNE2Z NPC cells and undifferentiated C666-1 cells, but not in well-differentiated CNE1 and immortalized NP-69 cells. Our findings suggest that high expression of UBE2C in human NPC is closely related to tumor malignancy, and may be a potential marker for NPC progression

  16. Microbial nitrilases: versatile, spiral forming, industrial enzymes.

    Science.gov (United States)

    Thuku, R N; Brady, D; Benedik, M J; Sewell, B T

    2009-03-01

    The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

  17. Catchment-scale conservation units identified for the threatened Yarra pygmy perch (Nannoperca obscura) in highly modified river systems.

    Science.gov (United States)

    Brauer, Chris J; Unmack, Peter J; Hammer, Michael P; Adams, Mark; Beheregaray, Luciano B

    2013-01-01

    Habitat fragmentation caused by human activities alters metapopulation dynamics and decreases biological connectivity through reduced migration and gene flow, leading to lowered levels of population genetic diversity and to local extinctions. The threatened Yarra pygmy perch, Nannoperca obscura, is a poor disperser found in small, isolated populations in wetlands and streams of southeastern Australia. Modifications to natural flow regimes in anthropogenically-impacted river systems have recently reduced the amount of habitat for this species and likely further limited its opportunity to disperse. We employed highly resolving microsatellite DNA markers to assess genetic variation, population structure and the spatial scale that dispersal takes place across the distribution of this freshwater fish and used this information to identify conservation units for management. The levels of genetic variation found for N. obscura are amongst the lowest reported for a fish species (mean heterozygosity of 0.318 and mean allelic richness of 1.92). We identified very strong population genetic structure, nil to little evidence of recent migration among demes and a minimum of 11 units for conservation management, hierarchically nested within four major genetic lineages. A combination of spatial analytical methods revealed hierarchical genetic structure corresponding with catchment boundaries and also demonstrated significant isolation by riverine distance. Our findings have implications for the national recovery plan of this species by demonstrating that N. obscura populations should be managed at a catchment level and highlighting the need to restore habitat and avoid further alteration of the natural hydrology.

  18. High Elevation Refugia for Bombus terricola (Hymenoptera: Apidae) Conservation and Wild Bees of the White Mountain National Forest.

    Science.gov (United States)

    Tucker, Erika M; Rehan, Sandra M

    2017-01-01

    Many wild bee species are in global decline, yet much is still unknown about their diversity and contemporary distributions. National parks and forests offer unique areas of refuge important for the conservation of rare and declining species populations. Here we present the results of the first biodiversity survey of the bee fauna in the White Mountain National Forest (WMNF). More than a thousand specimens were collected from pan and sweep samples representing 137 species. Three species were recorded for the first time in New England and an additional seven species were documented for the first time in the state of New Hampshire. Four introduced species were also observed in the specimens collected. A checklist of the species found in the WMNF, as well as those found previously in Strafford County, NH, is included with new state records and introduced species noted as well as a map of collecting locations. Of particular interest was the relatively high abundance of Bombus terricola Kirby 1837 found in many of the higher elevation collection sites and the single specimen documented of Bombus fervidus (Fabricius 1798). Both of these bumble bee species are known to have declining populations in the northeast and are categorized as vulnerable on the International Union for Conservation of Nature's Red List. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  19. Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Ji Qiongmei; Huang, C.-H.; Peng Jianbin; Hashmi, Sarwar; Ye Tianzhang; Chen Ying

    2007-01-01

    We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans

  20. Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide

    Directory of Open Access Journals (Sweden)

    Olga V. Kretova

    2017-09-01

    Full Text Available RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%–97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT, integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Keywords: HIV-1, RNAi targets, gene therapy, ultra-deep sequencing, conserved HIV-1 sequences

  1. Method of preparing highly active and thermostable preparations of liver uridin-kinase usable for enzymic synthesis of radioactive nucleoside-5'-phosphates

    International Nuclear Information System (INIS)

    Cihak, A.; Vesely, J.

    1975-01-01

    A method is described of preparing a high-activity uridine kinase for the enzymic synthesis of radioactive nucleoside-5m-phosphates of the pyrimidine series. The preparation is separated from male rat liver after intraperitoneal application of 5'-azacytidine. Examples are given showing detailed procedures for the conversion of uridine and 6-azauridine to the corresponding 5'-phosphates. (L.K.)

  2. High Re-Operation Rates Using Conserve Metal-On-Metal Total Hip Articulations

    DEFF Research Database (Denmark)

    Mogensen, S L; Jakobsen, Thomas; Christoffersen, Hardy

    2016-01-01

    INTRODUCTION: Metal-on-metal hip articulations have been intensely debated after reports of adverse reactions and high failure rates. The aim of this study was to retrospectively evaluate the implant of a metal-on.metal total hip articulation (MOM THA) from a single manufacture in a two-center st......INTRODUCTION: Metal-on-metal hip articulations have been intensely debated after reports of adverse reactions and high failure rates. The aim of this study was to retrospectively evaluate the implant of a metal-on.metal total hip articulation (MOM THA) from a single manufacture in a two...

  3. High spatial resolution mapping of land cover types in a priority area for conservation in the Brazilian savanna

    Science.gov (United States)

    Ribeiro, F.; Roberts, D. A.; Hess, L. L.; Davis, F. W.; Caylor, K. K.; Nackoney, J.; Antunes Daldegan, G.

    2017-12-01

    Savannas are heterogeneous landscapes consisting of highly mixed land cover types that lack clear distinct boundaries. The Brazilian Cerrado is a Neotropical savanna considered a biodiversity hotspot for conservation due to its biodiversity richness and rapid transformation of its landscape by crop and pasture activities. The Cerrado is one of the most threatened Brazilian biomes and only 2.2% of its original extent is strictly protected. Accurate mapping and monitoring of its ecosystems and adjacent land use are important to select areas for conservation and to improve our understanding of the dynamics in this biome. Land cover mapping of savannas is difficult due to spectral similarity between land cover types resulting from similar vegetation structure, floristically similar components, generalization of land cover classes, and heterogeneity usually expressed as small patch sizes within the natural landscape. These factors are the major contributor to misclassification and low map accuracies among remote sensing studies in savannas. Specific challenges to map the Cerrado's land cover types are related to the spectral similarity between classes of land use and natural vegetation, such as natural grassland vs. cultivated pasture, and forest ecosystem vs. crops. This study seeks to classify and evaluate the land cover patterns across an area ranked as having extremely high priority for future conservation in the Cerrado. The main objective of this study is to identify the representativeness of each vegetation type across the landscape using high to moderate spatial resolution imagery using an automated scheme. A combination of pixel-based and object-based approaches were tested using RapidEye 3A imagery (5m spatial resolution) to classify the Cerrado's major land cover types. The random forest classifier was used to map the major ecosystems present across the area, and demonstrated to have an effective result with 68% of overall accuracy. Post

  4. Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

    Directory of Open Access Journals (Sweden)

    Meuwissen Pieter J

    2012-04-01

    Full Text Available Abstract Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2 and non-canonical (B2 and C1422 HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2, the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we

  5. Ancient Exaptation of a CORE-SINE Retroposon into a Highly Conserved Mammalian Neuronal Enhancer of the Proopiomelanocortin Gene

    Science.gov (United States)

    Bumaschny, Viviana F; Low, Malcolm J; Rubinstein, Marcelo

    2007-01-01

    The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5′ distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution. PMID:17922573

  6. Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene.

    Directory of Open Access Journals (Sweden)

    Andrea M Santangelo

    2007-10-01

    Full Text Available The proopiomelanocortin gene (POMC is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5' distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution.

  7. Identification of immunogenic HLA-B7 "Achilles' heel" epitopes within highly conserved regions of HIV

    DEFF Research Database (Denmark)

    De Groot, Anne S; Rivera, Daniel S; McMurry, Julie A

    2008-01-01

    Genetic polymorphisms in class I human leukocyte antigen molecules (HLA) have been shown to determine susceptibility to HIV infection as well as the rate of progression to AIDS. In particular, the HLA-B7 supertype has been shown to be associated with high viral loads and rapid progression to dise...

  8. [Dynamic changes of soil microbial populations and enzyme activities in super-high yielding summer maize farmland soil].

    Science.gov (United States)

    Hou, Peng; Wang, Yong-jun; Wang, Kong-jun; Yang, Jin-sheng; Li, Deng-hai; Dong, Shu-ting; Liu, Jing-guo

    2008-08-01

    To reveal the characteristics of the dynamic changes of soil microbial populations and enzyme activities in super-high yielding ( > 15,000 kg x hm(-2)) summer maize farmland soil, a comparative study was conducted in the experimental fields in National Maize Engineering Research Center (Shandong). On the fields with an annual yield of >15,000 kg x hm(-2) in continuous three years, a plot with the yield of 20 322 kg x hm(-2) (HF) was chosen to make comparison with the conventional farmland (CF) whose maize yield was 8920. 1 kg x hm(-2). The numbers of bacteria, fungi, and actinomycetes as well as the activities of urease and invertase in 0-20 cm soil layer were determined. The results showed that in the growth period of maize, the numbers of bacteria, fungi, and actinomycetes in the two farmland soils increased first and declined then. At the later growth stages of maize, the numbers of soil microbes, especially those of bacteria and actinomycetes, were lower in HF than those in CF. At harvest stage, the ratio of the number of soil bacteria to fungi (B/ F) in HF was 2.03 times higher than that at sowing stage, and 3.02 times higher than that in CF. The B/F in CF had less difference at harvest and sowing stages. The soil urease activity in HF was significantly lower than that in CF at jointing stage, and the invertase activity in HF decreased rapidly after blooming stage, being significantly lower than that in CF.

  9. High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

    Science.gov (United States)

    Akif, Mohd; Georgiadis, Dimitris; Mahajan, Aman; Dive, Vincent; Sturrock, Edward D; Isaac, R Elwyn; Acharya, K Ravi

    2010-07-16

    Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 2010 Elsevier Ltd. All rights reserved.

  10. 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

    (chondroitin sulfate, fucoidan, xylan and pullulan) to determine the temperature-activity responses of hydrolysis of a related class of compounds. All 4 enzyme activities showed similarly low temperature optima in the range of 15 to 18degreesC. These temperature optima are considerably lower than most previous......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...... reports of temperature optima for enzyme activities in marine sediments. At 0degreesC, close to the in situ temperature, these enzyme activities achieved 13 to 38% of their rates at optimum temperatures. In one experiment, sulfate reduction rates were measured in parallel with extracellular enzymatic...

  11. A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies.

    Directory of Open Access Journals (Sweden)

    Cécile Fourrage

    Full Text Available Poc1 (Protein of Centriole 1 proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

  12. Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.

    Science.gov (United States)

    Cheng, Jian; Zhuang, Wei; Tang, Chenglun; Chen, Yong; Wu, Jinglan; Guo, Ting; Ying, Hanjie

    2017-03-01

    N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three resins: Amberzyme oxirane resin (AOR), poly (styrene-co-DVB)-Br resin (PBR) and amino resin (AR). The loading capacity and immobilized enzyme activity showed that AR was the best carrier. Three methods of glutaraldehyde cross-linking were tested and simultaneous cross-linking and immobilization was demonstrated to be the best method. The functional properties of immobilized AGE and NAL were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized AGE were obtained in 0.1 M potassium phosphate buffer at pH 7.5 and a temperature of 37 °C. Comparatively, the highest NAL activities were at pH 8.5. Meanwhile, an increase in K m (from 1.14 to 1.31 mg·mL -1 for AGE and from 1.05 to 1.25 mg·mL -1 for NAL) and a decrease in V max (from 177.53 to 106.37 µg·min -1 mL -1 for AGE and from 126.41 to 95.96 µg·min -1 mL -1 for NAL) were recorded after immobilization. The AR-glutaraldehyde-enzyme system exhibited better thermal stability than the free enzyme, and retained 72% of its initial activity even after eight repeated runs. The apparent activation energy (E a ) of the free and immobilized AGE (NAL) was 117.14 kJ·mol -1 (124.21 kJ·mol -1 ) and 78.45 kJ·mol -1 (66.64 kJ·mol -1 ), respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit. Subsequently, Neu5Ac production from GlcNAc using immobilized enzymes in one reactor was carried out resulting 101.45 g·L -1 of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.

  13. High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in Nicotiana tabacum

    Czech Academy of Sciences Publication Activity Database

    Synková, Helena; Semorádová, Šárka; Burketová, Lenka

    2004-01-01

    Roč. 79, č. 2 (2004), s. 169-179 ISSN 0167-6857 R&D Projects: GA ČR GA206/01/1061; GA ČR GA206/03/0310 Grant - others:Grantová agentura Univerzity Karlovy(CZ) 134/2001/B-Bio/PrF; Grantová agentura Univerzity Karlovy(CZ) Z5038910 Institutional research plan: CEZ:AV0Z5038910 Keywords : antioxidant enzymes * enzymes of intermediary metabolism * ex vitro Subject RIV: ED - Physiology Impact factor: 1.028, year: 2004

  14. Computation and Analysis of High Rocky Slope Safety in a Water Conservancy Project

    Directory of Open Access Journals (Sweden)

    Meng Yang

    2015-01-01

    Full Text Available An integrated method, covering the actual monitoring analysis, practical geological model, and theoretical mathematical simulation model, is systematically proposed and successfully applied. Deformation characteristic of a unique high rocky slope was firstly analyzed from multiple angles and multiple layers by changeable elevations and distances. Arrangements of monitoring points were listed and monitoring equipment was designed to comprise a complete monitoring system. Present larger displacement was concluded for bottom larger displacement caused by water erosion and middle larger displacement formed by seepage. Temporal and spatial displacements rule study of multiple-points linkage effects with water factor proved this conclusion. To better excavate useful message and analyze the deep rule from the practical monitoring data, the slope geological model was conducted and rock mechanic parameters were researched. Finally, a unique three-dimensional finite element model was applied to approach the structure character using numerical simulations. The corresponding strength criterion was used to determine the safety coefficient by selecting a typical section. Subsequently, an integrated three-dimensional finite element model of the slope and dam was developed and more detailed deformation evolution mechanism was revealed. This study is expected to provide a powerful and systematic method to analyze very high, important, and dangerous slopes.

  15. High seroprevalence of Mycoplasma pneumoniae IgM in acute Q fever by enzyme-linked immunosorbent assay (ELISA.

    Directory of Open Access Journals (Sweden)

    Chung-Hsu Lai

    Full Text Available Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA, a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001 and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001 of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5% and seroconversion rate (33.3% of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases with those who were negative (43 cases, the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255, sore throat (8.5% vs. 16.3%, p=0.351, cough (35.6% vs. 23.3%, p=0.199, and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258, were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

  16. Constrained instanton and baryon number non-conservation at high energies

    International Nuclear Information System (INIS)

    Sil'vestrov, P.G.

    1992-01-01

    The main subject of this paper is the calculation of corrections to instanton action ΔS∼(mρ) 4 log(mρ)/g 2 (ρ is the intanton radius) in the SU(2) Yang-Mills theory. The total cross section for baryon number violating processes at high energies is usually parametrized as σ tat ∝exp(4π/αF(ε)), where α=g 2 /4π, ε=√s/E 0 , E 0 =√6πm w /α. In the present paper the third nontrivial term of the F(ε) expansion is obtained. The unknown correction to F(ε) are expected to be of the order of ε 8/3 . The total cross section is extremely sensitive to the value of single instanton action. For sufficiently heavy Higgs boson the ρ-dependent part of the instanton action is changed drastically. 21 refs.; 1 fig

  17. Enzyme-treated wheat bran alters gut microbiota and liver metabolome in mice fed a high fat diet

    Science.gov (United States)

    Enzyme-treated wheat bran (ETWB) is a fermentable dietary fiber that has been shown to decrease body fat and modify the gut microbiome. However, it is not clear how these microbiome changes impact peripheral tissue metabolism. We hypothesized that supplementation with ETWB would change gut-derived...

  18. Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.

    2017-01-01

    Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obta...

  19. The Probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme

    NARCIS (Netherlands)

    Anwar, Munir A.; Kralj, Slavko; van der Maarel, Marc J. E. C.; Dijkhuizen, Lubbert

    Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a

  20. Mesoscopic dynamics of diffusion-influenced enzyme kinetics.

    Science.gov (United States)

    Chen, Jiang-Xing; Kapral, Raymond

    2011-01-28

    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t(-1/2) and t(-3/2) power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  1. Mesoscopic dynamics of diffusion-influenced enzyme kinetics

    Science.gov (United States)

    Chen, Jiang-Xing; Kapral, Raymond

    2011-01-01

    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t^{-1/2} and t^{-3/2} power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  2. Mice selectively bred for high voluntary wheel-running behavior conserve more fat despite increased exercise.

    Science.gov (United States)

    Hiramatsu, Layla; Garland, Theodore

    2018-04-20

    Physical activity is an important component of energy expenditure, and acute changes in activity can lead to energy imbalances that affect body composition, even under ad libitum food availability. One example of acute increases in physical activity is four replicate, selectively-bred High Runner (HR) lines of mice that voluntarily run ~3-fold more wheel revolutions per day over 6-day trials and are leaner, as compared with four non-selected control (C) lines. We expected that voluntary exercise would increase food consumption, build lean mass, and reduce fat mass, but that these effects would likely differ between HR and C lines or between the sexes. We compared wheel running, cage activity, food consumption, and body composition between HR and C lines for young adults of both sexes, and examined interrelationships of those traits across 6 days of wheel access. Before wheel testing, HR mice weighed less than C, primarily due to reduced lean mass, and females were lighter than males, entirely due to lower lean mass. Over 6 days of wheel access, all groups tended to gain small amounts of lean mass, but lose fat mass. HR mice lost less fat than C mice, in spite of much higher activity levels, resulting in convergence to a fat mass of ~1.7 g for all 4 groups. HR mice consumed more food than C mice (with body mass as a covariate), even accounting for their higher activity levels. No significant sex-by-linetype interactions were observed for any of the foregoing traits. Structural equation models showed that the four sex-by-linetype groups differed considerably in the complex phenotypic architecture of these traits. Interrelationships among traits differed by genetic background and sex, lending support to the idea that recommendations regarding weight management, diet, and exercise may need to be tailored to the individual level. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein

    Directory of Open Access Journals (Sweden)

    Gibbons I R

    2002-07-01

    Full Text Available Abstract Background The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p, an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. Results Midasin is present as a single-copy gene encoding a well-conserved protein of ~600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa. Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa, followed by an AAA domain containing six tandem AAA protomers (~30 kDa each, a linker domain (260 kDa, an acidic domain (~70 kDa containing 35–40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. Conclusions The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site.

  4. Toxicity and cosmetic result of partial breast high-dose-rate interstitial brachytherapy for conservatively operated early breast cancer

    International Nuclear Information System (INIS)

    Xiu Xia; Tripuraneni Prabhakar; Giap Huan; Lin Ray; Chu Colin

    2007-01-01

    Objective: Objective To study the method, side effects and cosmetic outcome of high- dose-rate (HDR) accelerated partial breast interstitial irradiation (APBI) alone in early stage breast cancer' after conservative surgery. Methods: From February 2002 to June 2003,47 breast cancer lesions from 46 patients suffering from stage I/II breast cancer were treated with HDR 192 Ir APBI after conservative surgery. All patients were over 40 year-old, with T1-2N0-1 (≤3 lymph nodes positive), surgical margin > 1-2 mm, but those having lobular or inflammatory breast cancer were excluded. HDR brachytherapy with 34 Gy, 10 fractions/5 days was used after surgery, toxic reaction and cosmetic outcome were observed in one month, 6 and 12 months respectively. Results: Follow up of 1846 months, 34 months was carried out for the whole group. During the treatment, acute reactions including: erythema, edema, tenderness and infection, all under I-II grade, none of III-IV grade were observed in 21 patients(46%); late toxicity reactions: skin fibrosis, breast tenderness, fat necrosis, and telangiectasia, totally 20 patients (43%) were observed: 2 patients in III grade but one patient received 6 cycle chemotherapy. The result of cosmetic outcome evaluation was excellent or good, at 6 months 95% and 12 months 98%, respectively, but there was no recurfence. Conclusions: Excellent and favorable cosmetic results are noted after APBI by interstitial alone. Acute and late reactions are few. Long term observation is necessary for the rate of' local control. (authors)

  5. Response to angiotensin-converting enzyme inhibition is selectively blunted by high sodium in angiotensin-converting enzyme DD genotype: evidence for gene-environment interaction in healthy volunteers.

    Science.gov (United States)

    Lely, A Titia; Heerspink, Hiddo J Lambers; Zuurman, Mike; Visser, Folkert W; Kocks, Menno J A; Boomsma, Frans; Navis, Gerjan

    2010-12-01

    Renin-angiotensin-aldosterone system blockade is a cornerstone in cardiovascular protection. Angiotensin-converting enzyme (ACE)-DD genotype has been associated with resistance to angiotensin-converting enzyme inhibition (ACEi), but data are conflicting. As sodium intake modifies the effect of ACEi as well as the genotype-phenotype relationship, we hypothesize gene-environment interaction between sodium-status, the response to ACEi, and ACE genotype. Thirty-five male volunteers (26 ± 9 years; II n = 6, ID n = 18, DD n = 11) were studied during placebo and ACEi (double blind, enalapril 20 mg/day) on low [7 days 50 mmol Na/day (low salt)] and high [7 days 200 mmol Na/day (high salt)] sodium, with a washout of 6 weeks in-between. After each period mean arterial pressure (MAP) was measured before and during graded infusion of angiotensin II (Ang II). During high salt, ACEi reduced MAP in II and ID, but not in DD [II: 88 (78-94) versus 76 (72-88); ID: 87 (84-91) versus 83 (79-87); both P DD: 86 (82-96) versus 88 (80-90); ns, P DD: 84 (80-91) versus 81 (75-85); all P DD, with an 18% rise in MAP during the highest dose versus 22 and 31% in ID and II (P DD genotype during high salt, accompanied by blunted sensitivity to Ang II. Low salt corrects both abnormalities. Further analysis of this gene-environment interaction in patients may contribute to strategies for improvement of individual treatment efficacy.

  6. A simpler sampling interface of venturi easy ambient sonic-spray ionization mass spectrometry for high-throughput screening enzyme inhibitors.

    Science.gov (United States)

    Liu, Ning; Liu, Yang; Yang, YuHan; He, Lan; Ouyang, Jin

    2016-03-24

    High-throughput screening (HTS) is often required in enzyme inhibitor drugs screening. Mass spectrometry (MS) provides a powerful method for high-throughput screening enzyme inhibitors because its high speed, sensitivity and property of lable free. However, most of the MS methods need complicated sampling interface system. Overall throughput was limited by sample loading in these cases. In this study, we develop a simple interface which coupled droplet segmented system to a venturi easy ambient sonic-spray ionization mass spectrometer. It is fabricated by using a single capillary to act as both sampling probe and the emitter, which simplifies the construction, reduces the cost and shorten the sampling time. Samples sucked by venturi effect are segmented to nanoliter plugs by air, then the plugs can be detected by MS directly. This system eliminated the need for flow injection which was popular used in classic scheme. The new system is applied to screen angiotensin converting enzyme inhibitors. High-throughput was achieved in analyzing 96 samples at 1.6 s per sample. The plugs formation was at 0.5s per sample. Carry-over between samples was less than 5%, the peak height RSD was 2.92% (n = 15). Dose-response curves of 3 known inhibitors were also measured to validate its potential in drug discovery. The calculated IC50 agreed well with reported values. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

    Directory of Open Access Journals (Sweden)

    K. M. Singh

    2014-01-01

    Full Text Available The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs. We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs, carbohydrate binding module (CBM: 23 contigs, glycosyl transferase (GT: 373 contigs, carbohydrate esterases (CE: 259 contigs, and polysaccharide lyases (PE: 16 contigs. The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

  8. Synthesis and discovery of highly functionalized mono- and bis-spiro-pyrrolidines as potent cholinesterase enzyme inhibitors.

    Science.gov (United States)

    Kia, Yalda; Osman, Hasnah; Suresh Kumar, Raju; Basiri, Alireza; Murugaiyah, Vikneswaran

    2014-04-01

    Novel mono and bis spiropyrrolidine derivatives were synthesized via an efficient ionic liquid mediated, 1,3-dipolar cycloaddition methodology and evaluated in vitro for their AChE and BChE inhibitory activities in search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable AChE inhibitory activities with IC50 values ranging from 1.68 to 21.85 μM, wherein compounds 8d and 8j were found to be most active inhibitors against AChE and BChE with IC50 values of 1.68 and 2.75 μM, respectively. Molecular modeling simulation on Torpedo californica AChE and human BChE receptors, showed good correlation between IC50 values and binding interaction template of the most active inhibitors docked into the active site of their relevant enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Non-surgical breast-conservation treatment (KORTUC-BCT) using a new image-guided, enzyme-targeted, and breast cancer stem cell targeted radiosensitization treatment (KORTUC II) for patients with stage I or II breast cancer

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Kubota, Kei; Tadokoro, Michiko

    2012-01-01

    Tumor tissue can be re-oxygenated by inactivating peroxidase/catalase in the tumor tissue through application of hydrogen peroxide. The hydrogen peroxide in turn is then degraded to produce oxygen. In this way, low-LET (linear energy transfer) radioresistant tumors can be transformed into radiosensitive ones (Ogawa Y, et al: Int J Mol Med 12: 453-458, 845-850, 2003, Ogawa Y, et al: Int J Mol Med 14: 397-403, 2004, Kariya S, et al: Int J Radiat Oncol Biol Phys 75: 449-454, 2009). The purpose of the present study was to establish a non-surgical breast-conservation treatment (KORTUC-BCT) by utilizing a novel Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas, Type II (KORTUC II) radiosensitization treatment. KORTUC I was shown to remarkably enhance radiotherapeutic effects in various types of superficially exposed and locally advanced neoplasms (Ogawa Y, et al: Oncol Rep 19: 1389-1394, 2008). Based on clinical experiences using KORTUC I, a new radiosensitizing agent containing hydrogen peroxide and sodium hyaluronate has been developed for intra-tumoral injection in various tumors which are not superficially exposed. The agent is composed of 0.5% hydrogen peroxide and 0.83% sodium hyaluronate (CD44 molecule). Sodium hyaluronate mixed with hydrogen peroxide attaches to CD44-positive tumor cells, which are generally reported to be breast cancer stem cells. This new method, named KORTUC II, was approved by our local ethics committee for treatment of advanced skin cancer (including malignant melanoma), bone/soft tissue malignant neoplasm, breast cancer and metastatic lymph node. A total of 39 early stage breast cancer patients (stage I: 12 patients and stage II: 27) were enrolled in the KORTUC II trial upon fully informed consent. Mean age of the patients was 61.1 years old. All 39 patients were unable or unwilling to undergo surgery and therefore undertook non-surgical breast-conservation treatment (KORTUC-BCT) by KORTUC II. A maximum of 6 ml of the agent was

  10. Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

    Science.gov (United States)

    Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry A F; Rothman, Alan L; Mathew, Anuja

    2014-01-01

    Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation. © 2013 John Wiley & Sons Ltd.

  11. A Web-Based GIS for Reporting Water Usage in the High Plains Underground Water Conservation District

    Science.gov (United States)

    Jia, M.; Deeds, N.; Winckler, M.

    2012-12-01

    The High Plains Underground Water Conservation District (HPWD) is the largest and oldest of the Texas water conservation districts, and oversees approximately 1.7 million irrigated acres. Recent rule changes have motivated HPWD to develop a more automated system to allow owners and operators to report well locations, meter locations, meter readings, the association between meters and wells, and contiguous acres. INTERA, Inc. has developed a web-based interactive system for HPWD water users to report water usage and for the district to better manage its water resources. The HPWD web management system utilizes state-of-the-art GIS techniques, including cloud-based Amazon EC2 virtual machine, ArcGIS Server, ArcSDE and ArcGIS Viewer for Flex, to support web-based water use management. The system enables users to navigate to their area of interest using a well-established base-map and perform a variety of operations and inquiries against their spatial features. The application currently has six components: user privilege management, property management, water meter registration, area registration, meter-well association and water use report. The system is composed of two main databases: spatial database and non-spatial database. With the help of Adobe Flex application at the front end and ArcGIS Server as the middle-ware, the spatial feature geometry and attributes update will be reflected immediately in the back end. As a result, property owners, along with the HPWD staff, collaborate together to weave the fabric of the spatial database. Interactions between the spatial and non-spatial databases are established by Windows Communication Foundation (WCF) services to record water-use report, user-property associations, owner-area associations, as well as meter-well associations. Mobile capabilities will be enabled in the near future for field workers to collect data and synchronize them to the spatial database. The entire solution is built on a highly scalable cloud

  12. Conservation Value

    OpenAIRE

    Tisdell, Clement A.

    2010-01-01

    This paper outlines the significance of the concept of conservation value and discusses ways in which it is determined paying attention to views stemming from utilitarian ethics and from deontological ethics. The importance of user costs in relation to economic decisions about the conservation and use of natural resources is emphasised. Particular attention is given to competing views about the importance of conserving natural resources in order to achieve economic sustainability. This then l...

  13. Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

    Science.gov (United States)

    Ahmed, Nisar; Tetlow, Ian J; Nawaz, Sehar; Iqbal, Ahsan; Mubin, Muhammad; Nawaz ul Rehman, Muhammad Shah; Butt, Aisha; Lightfoot, David A; Maekawa, Masahiko

    2015-08-30

    High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis. © 2014 Society of Chemical Industry.

  14. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel

    2017-01-01

    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

  15. Loss of a highly conserved sterile alpha motif domain gene (WEEP) results in pendulous branch growth in peach trees.

    Science.gov (United States)

    Hollender, Courtney A; Pascal, Thierry; Tabb, Amy; Hadiarto, Toto; Srinivasan, Chinnathambi; Wang, Wanpeng; Liu, Zhongchi; Scorza, Ralph; Dardick, Chris

    2018-05-15

    Plant shoots typically grow upward in opposition to the pull of gravity. However, exceptions exist throughout the plant kingdom. Most conspicuous are trees with weeping or pendulous branches. While such trees have long been cultivated and appreciated for their ornamental value, the molecular basis behind the weeping habit is not known. Here, we characterized a weeping tree phenotype in Prunus persica (peach) and identified the underlying genetic mutation using a genomic sequencing approach. Weeping peach tree shoots exhibited a downward elliptical growth pattern and did not exhibit an upward bending in response to 90° reorientation. The causative allele was found to be an uncharacterized gene, Ppa013325 , having a 1.8-Kb deletion spanning the 5' end. This gene, dubbed WEEP , was predominantly expressed in phloem tissues and encodes a highly conserved 129-amino acid protein containing a sterile alpha motif (SAM) domain. Silencing WEEP in the related tree species Prunus domestica (plum) resulted in more outward, downward, and wandering shoot orientations compared to standard trees, supporting a role for WEEP in directing lateral shoot growth in trees. This previously unknown regulator of branch orientation, which may also be a regulator of gravity perception or response, provides insights into our understanding of how tree branches grow in opposition to gravity and could serve as a critical target for manipulating tree architecture for improved tree shape in agricultural and horticulture applications. Copyright © 2018 the Author(s). Published by PNAS.

  16. Neuronal patterning of the tubular collar cord is highly conserved among enteropneusts but dissimilar to the chordate neural tube.

    Science.gov (United States)

    Kaul-Strehlow, Sabrina; Urata, Makoto; Praher, Daniela; Wanninger, Andreas

    2017-08-01

    A tubular nervous system is present in the deuterostome groups Chordata (cephalochordates, tunicates, vertebrates) and in the non-chordate Enteropneusta. However, the worm-shaped enteropneusts possess a less complex nervous system featuring only a short hollow neural tube, whereby homology to its chordate counterpart remains elusive. Since the majority of data on enteropneusts stem from the harrimaniid Saccoglossus kowalevskii, putative interspecific variations remain undetected resulting in an unreliable ground pattern that impedes homology assessments. In order to complement the missing data from another enteropneust family, we investigated expression of key neuronal patterning genes in the ptychoderid Balanoglossus misakiensis. The collar cord of B. misakiensis shows anterior Six3/6 and posterior Otx + Engrailed expression, in a region corresponding to the chordate brain. Neuronal Nk2.1/Nk2.2 expression is absent. Interestingly, we found median Dlx and lateral Pax6 expression domains, i.e., a condition that is reversed compared to chordates. Comparative analyses reveal that adult nervous system patterning is highly conserved among the enteropneust families Harrimaniidae, Spengelidae and Ptychoderidae. BmiDlx and BmiPax6 have no corresponding expression domains in the chordate brain, which may be indicative of independent acquisition of a tubular nervous system in Enteropneusta and Chordata.

  17. Assessment of some high-order finite difference schemes on the scalar conservation law with periodical conditions

    Directory of Open Access Journals (Sweden)

    Alina BOGOI

    2016-12-01

    Full Text Available Supersonic/hypersonic flows with strong shocks need special treatment in Computational Fluid Dynamics (CFD in order to accurately capture the discontinuity location and his magnitude. To avoid numerical instabilities in the presence of discontinuities, the numerical schemes must generate low dissipation and low dispersion error. Consequently, the algorithms used to calculate the time and space-derivatives, should exhibit a low amplitude and phase error. This paper focuses on the comparison of the numerical results obtained by simulations with some high resolution numerical schemes applied on linear and non-linear one-dimensional conservation low. The analytical solutions are provided for all benchmark tests considering smooth periodical conditions. All the schemes converge to the proper weak solution for linear flux and smooth initial conditions. However, when the flux is non-linear, the discontinuities may develop from smooth initial conditions and the shock must be correctly captured. All the schemes accurately identify the shock position, with the price of the numerical oscillation in the vicinity of the sudden variation. We believe that the identification of this pure numerical behavior, without physical relevance, in 1D case is extremely useful to avoid problems related to the stability and convergence of the solution in the general 3D case.

  18. The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

    Directory of Open Access Journals (Sweden)

    Suneeth F Mathew

    Full Text Available HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon' contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

  19. The implications of RCRA [Resource Conservation and Recovery Act] regulation for the disposal of transuranic and high-level waste

    International Nuclear Information System (INIS)

    Sigmon, C.F.; Sharples, F.E.; Smith, E.D.

    1988-01-01

    In May of 1987 the Department of Energy (DOE) published a rule interpreting the definition of ''byproduct'' under the Atomic Energy Act. This byproduct rule clarified the role of the Resource Conservation and Recovery Act (RCRA) in the regulation of DOE's radioactive waste management activities. According to the rule, only the radioactive portion of DOE's mixed radioactive and hazardous waste (mixed waste), including mixed transuranic (TRU) and high-level waste (HLW), is exempt from RCRA under the byproduct exemption. The portion of a waste that is hazardous as defined by RCRA is subject to full regulation under RCRA. Because the radioactive and hazardous portions of m any, if not most, DOE wastes are likely to be inseparable, the rule in effect makes most mixed wastes subject to dual regulation. The potential application of RCRA to facilities such as the Waste Isolation Pilot Plant (WIPP) and the HLW repository creates unique challenges for both the DOE and regulatory authorities. Strategies must be developed to assure compliance with RCRA without either causing excessive administrative burdens or abandoning the goal of minimizing radiation exposure. This paper will explore some of the potential regulatory options for and recent trends in the regulation of TRU and HLW under RCRA

  20. Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

    Directory of Open Access Journals (Sweden)

    Solbak Sara MØ

    2011-02-01

    Full Text Available Abstract Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L- domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX, is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively

  1. Enzyme-Initiated Quinone-Chitosan Conjugation Chemistry: Toward A General in Situ Strategy for High-Throughput Photoelectrochemical Enzymatic Bioanalysis.

    Science.gov (United States)

    Wang, Guang-Li; Yuan, Fang; Gu, Tiantian; Dong, Yuming; Wang, Qian; Zhao, Wei-Wei

    2018-02-06

    Herein we report a general and novel strategy for high-throughput photoelectrochemical (PEC) enzymatic bioanalysis on the basis of enzyme-initiated quinone-chitosan conjugation chemistry (QCCC). Specifically, the strategy was illustrated by using a model quinones-generating oxidase of tyrosinase (Tyr) to catalytically produce 1,2-bezoquinone or its derivative, which can easily and selectively be conjugated onto the surface of the chitosan deposited PbS/NiO/FTO photocathode via the QCCC. Upon illumination, the covalently attached quinones could act as electron acceptors of PbS quantum dots (QDs), improving the photocurrent generation and thus allowing the elegant probing of Tyr activity. Enzyme cascades, such as alkaline phosphatase (ALP)/Tyr and β-galactosidase (Gal)/Tyr, were further introduced into the system for the successful probing of the corresponding targets. This work features not only the first use of QCCC in PEC bioanalysis but also the separation of enzymatic reaction from the photoelectrode as well as the direct signal recording in a split-type protocol, which enables quite convenient and high-throughput detection as compared to previous formats. More importantly, by using numerous other oxidoreductases that involve quinones as reactants/products, this protocol could serve as a common basis for the development of a new class of QCCC-based PEC enzymatic bioanalysis and further extended for general enzyme-labeled PEC bioanalysis of versatile targets.

  2. Reversible tetramerization of human TK1 to the high catalytic efficient form is induced by pyrophosphate, in addition to tripolyphosphates, or high enzyme concentration

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2009-01-01

    of ATP is necessary for tetramerisation and how the reaction velocity is influenced by the enzyme concentration. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerisation, and that kinetics and tetramerisation are closely related. Furthermore, enzyme...... concentration was found to have a pivotal effect on catalytic efficiency.......Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism catalyzing the first step in the synthesis of dTTP by the transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5´-hydroxyl group of thymidine forming dTMP. Human TK1 is cytosolic...

  3. High conservation level of CD8(+) T cell immunogenic regions within an unusual H1N2 human influenza variant.

    Science.gov (United States)

    Komadina, Naomi; Quiñones-Parra, Sergio M; Kedzierska, Katherine; McCaw, James M; Kelso, Anne; Leder, Karin; McVernon, Jodie

    2016-10-01

    Current seasonal influenza vaccines require regular updates due to antigenic drift causing loss of effectiveness and therefore providing little or no protection against novel influenza A subtypes. Next generation vaccines capable of eliciting CD8(+) T cell (CTL) mediated cross-protective immunity may offer a long-term alternative strategy. However, measuring pre- and existing levels of CTL cross-protection in humans is confounded by differences in infection histories across individuals. During 2000-2003, H1N2 viruses circulated persistently in the human population for the first time and we hypothesized that the viral nucleoprotein (NP) contained novel CTL epitopes that may have contributed to the survival of the viruses. This study describes the immunogenic NP peptides of H1N1, H2N2, and H3N2 influenza viruses isolated from humans over the past century, 1918-2003, by comparing this historical dataset to reference NP peptides from H1N2 that circulated in humans during 2000-2003. Observed peptides sequences ranged from highly conserved (15%) to highly variable (12%), with variation unrelated to reported immunodominance. No unique NP peptides which were exclusive to the H1N2 viruses were noted. However, the virus had inherited the NP from a recently emerged H3N2 variant containing novel peptides, which may have assisted its persistence. Any advantage due to this novelty was subsequently lost with emergence of a newer H3N2 variant in 2003. Our approach has potential to provide insight into the population context in which influenza viruses emerge, and may help to inform immunogenic peptide selection for CTL-inducing influenza vaccines. J. Med. Virol. 88:1725-1732, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Use of enzymes to minimize the rheological dough problems caused by high levels of damaged starch in starch-gluten systems.

    Science.gov (United States)

    Barrera, Gabriela N; León, Alberto E; Ribotta, Pablo D

    2016-05-01

    During wheat milling, starch granules can experience mechanical damage, producing damaged starch. High levels of damaged starch modify the physicochemical properties of wheat flour, negatively affecting the dough behavior as well as the flour quality and cookie and bread making quality. The aim of this work was to evaluate the effect of α-amylase, maltogenic amylase and amyloglucosidase on dough rheology in order to propose alternatives to reduce the issues related to high levels of damaged starch. The dough with a high level of damaged starch became more viscous and resistant to deformations as well as less elastic and extensible. The soluble fraction of the doughs influenced the rheological behavior of the systems. The α-amylase and amyloglucosidase reduced the negative effects of high damaged starch contents, improving the dough rheological properties modified by damaged starch. The rheological behavior of dough with the higher damaged-starch content was related to a more open gluten network arrangement as a result of the large size of the swollen damaged starch granules. We can conclude that the dough rheological properties of systems with high damaged starch content changed positively as a result of enzyme action, particularly α-amylase and amyloglucosidase additions, allowing the use of these amylases and mixtures of them as corrective additives. Little information was reported about amyloglucosidase activity alone or combined with α-amylase. The combinations of these two enzymes are promising to minimize the negative effects caused by high levels of damaged starch on product quality. More research needs to be done on bread quality combining these two enzymes. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  5. Surgical versus conservative treatment for high-risk stress fractures of the lower leg (anterior tibial cortex, navicular and fifth metatarsal base): a systematic review

    NARCIS (Netherlands)

    Mallee, W.H.; Weel, H.; van Dijk, C.N.; van Tulder, M.W.; Kerkhoffs, G.M.; Lin, C.W.C.

    2015-01-01

    Aim To compare surgical and conservative treatment for high-risk stress fractures of the anterior tibial cortex, navicular and proximal fifth metatarsal. Methods Systematic searches of CENTRAL, MEDLINE, EMBASE, CINAHL, SPORTDiscus and PEDro were performed to identify relevant prospective and

  6. Comparing the cost-effectiveness of water conservation policies in a depleting aquifer:A dynamic analysis of the Kansas High Plains

    Science.gov (United States)

    This research analyzes two groundwater conservation policies in the Kansas High Plains located within the Ogallala aquifer: 1) cost-share assistance to increase irrigation efficiency; and 2) incentive payments to convert irrigated crop production to dryland crop production. To compare the cost-effec...

  7. Spatially conserved regulatory elements identified within human and mouse Cd247 gene using high-throughput sequencing data from the ENCODE project

    DEFF Research Database (Denmark)

    Pundhir, Sachin; Hannibal, Tine Dahlbæk; Bang-Berthelsen, Claus Heiner

    2014-01-01

    . In this study, we have utilized the wealth of high-throughput sequencing data produced during the Encyclopedia of DNA Elements (ENCODE) project to identify spatially conserved regulatory elements within the Cd247 gene from human and mouse. We show the presence of two transcription factor binding sites...

  8. Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

    Science.gov (United States)

    Sharma, S; Tyagi, R; Gupta, M N; Singh, T P

    2001-01-01

    For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar

  9. A Resource Conservation Unit.

    Science.gov (United States)

    Porter, Philip D.

    1979-01-01

    Describes a variety of learning activities for teaching elementary and junior high students about air, water, and energy conservation techniques. Suggests community resources, social studies objectives, language skills, and 20 activities. (CK)

  10. 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...

  11. Synthesis and biological studies of highly concentrated lisinopril-capped gold nanoparticles for CT tracking of angiotensin converting enzyme (ACE)

    Science.gov (United States)

    Ghann, William E.; Aras, Omer; Fleiter, Thorsten; Daniel, Marie-Christine

    2011-05-01

    For patients with a history of heart attack or stroke, the prevention of another cardiovascular or cerebrovascular event is crucial. The development of cardiac and pulmonary fibrosis has been associated with overexpression of tissue angiotensin-converting enzyme (ACE). Recently, gold nanoparticles (GNPs) have shown great potential as X-ray computed tomography (CT) contrast agents. Since lisinopril is an ACE inhibitor, it has been used as coating on GNPs for targeted imaging of tissue ACE in prevention of fibrosis. Herein, lisinopril-capped gold nanoparticles (LIS-GNPs) were synthesized up to a concentration of 55 mgAu/mL. Their contrast was measured using CT and the results were compared to Omnipaque, a commonly used iodine-based contrast agent. The targeting ability of these LIS-GNPs was also assessed.

  12. The effect of different enzymes on the quality of high-fibre enriched brewer's spent grain breads.

    Science.gov (United States)

    Stojceska, Valentina; Ainsworth, Paul

    2008-10-15

    The brewing industry produces large quantities of waste co-products. There is increasing pressure to ensure total utilisation of such products to address economic and environmental concerns. Brewer's spent grain (BSG) the main by-product of the brewing industry is rich in dietary fibre and has a strong potential to be recycled. The overall objective of this study was to incorporate BSG into wheat flour breads together with a range of different enzymes (Maxlife 85, Lipopan Extra, Pentopan Mono BG and Celluclast) and evaluate the bread quality. A number of nutritional and textural properties of the finished product were studied. The incorporation of BSG significantly (Pbreads containing 0%, 10%, 20% and 30% BSG). Image analysis of the bread structure obtained from the C-cell analyzer showed that the most significantly (P<0.001) open network was obtained using LE, followed by PE and PCE. Copyright © 2008 Elsevier Ltd. All rights reserved.

  13. Model My Watershed: A high-performance cloud application for public engagement, watershed modeling and conservation decision support

    Science.gov (United States)

    Aufdenkampe, A. K.; Tarboton, D. G.; Horsburgh, J. S.; Mayorga, E.; McFarland, M.; Robbins, A.; Haag, S.; Shokoufandeh, A.; Evans, B. M.; Arscott, D. B.

    2017-12-01

    The Model My Watershed Web app (https://app.wikiwatershed.org/) and the BiG-CZ Data Portal (http://portal.bigcz.org/) and are web applications that share a common codebase and a common goal to deliver high-performance discovery, visualization and analysis of geospatial data in an intuitive user interface in web browser. Model My Watershed (MMW) was designed as a decision support system for watershed conservation implementation. BiG CZ Data Portal was designed to provide context and background data for research sites. Users begin by creating an Area of Interest, via an automated watershed delineation tool, a free draw tool, selection of a predefined area such as a county or USGS Hydrological Unit (HUC), or uploading a custom polygon. Both Web apps visualize and provide summary statistics of land use, soil groups, streams, climate and other geospatial information. MMW then allows users to run a watershed model to simulate different scenarios of human impacts on stormwater runoff and water-quality. BiG CZ Data Portal allows users to search for scientific and monitoring data within the Area of Interest, which also serves as a prototype for the upcoming Monitor My Watershed web app. Both systems integrate with CUAHSI cyberinfrastructure, including visualizing observational data from CUAHSI Water Data Center and storing user data via CUAHSI HydroShare. Both systems also integrate with the new EnviroDIY Water Quality Data Portal (http://data.envirodiy.org/), a system for crowd-sourcing environmental monitoring data using open-source sensor stations (http://envirodiy.org/mayfly/) and based on the Observations Data Model v2.

  14. Functional Comparison for Lipid Metabolism and Intestinal and Fecal Microflora Enzyme Activities between Low Molecular Weight Chitosan and Chitosan Oligosaccharide in High-Fat-Diet-Fed Rats.

    Science.gov (United States)

    Chiu, Chen-Yuan; Feng, Shih-An; Liu, Shing-Hwa; Chiang, Meng-Tsan

    2017-07-24

    The present study investigated and compared the regulatory effects on the lipid-related metabolism and intestinal disaccharidase/fecal bacterial enzyme activities between low molecular weight chitosan and chitosan oligosaccharide in high-fat-diet-fed rats. Diet supplementation of low molecular weight chitosan showed greater efficiency than chitosan oligosaccharide in suppressing the increased weights in body and in liver and adipose tissues of high-fat-diet-fed rats. Supplementation of low molecular weight chitosan also showed a greater improvement than chitosan oligosaccharide in imbalance of plasma, hepatic, and fecal lipid profiles, and intestinal disaccharidase activities in high-fat-diet-fed rats. Moreover, both low molecular weight chitosan and chitosan oligosaccharide significantly decreased the fecal microflora mucinase and β-glucuronidase activities in high-fat-diet-fed rats. These results suggest that low molecular weight chitosan exerts a greater positive improvement than chitosan oligosaccharide in lipid metabolism and intestinal disaccharidase activity in high-fat-diet-induced obese rats.

  15. A highly sensitive electrochemical biosensor for catechol using conducting polymer reduced graphene oxide-metal oxide enzyme modified electrode.

    Science.gov (United States)

    Sethuraman, V; Muthuraja, P; Anandha Raj, J; Manisankar, P

    2016-10-15

    The fabrication, characterization and analytical performances were investigated for a catechol biosensor, based on the PEDOT-rGO-Fe2O3-PPO composite modified glassy carbon (GC) electrode. The graphene oxide (GO) doped conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) was prepared through electrochemical polymerization by potential cycling. Reduction of PEDOT-GO was carried out by amperometric method. Fe2O3 nanoparticles were synthesized in ethanol by hydrothermal method. The mixture of Fe2O3, PPO and glutaraldehyde was casted on the PEDOT-rGO electrode. The surface morphology of the modified electrodes was studied by FE-SEM and AFM. Cyclic voltammetric studies of catechol on the enzyme modified electrode revealed higher reduction peak current. Determination of catechol was carried out successfully by Differential Pulse Voltammetry (DPV) technique. The fabricated biosensor investigated shows a maximum current response at pH 6.5. The catechol biosensor exhibited wide sensing linear range from 4×10(-8) to 6.20×10(-5)M, lower detection limit of 7×10(-9)M, current maxima (Imax) of 92.55µA and Michaelis-Menten (Km) constant of 30.48µM. The activation energy (Ea) of enzyme electrode is 35.93KJmol(-1) at 50°C. There is no interference from d-glucose and l-glutamic acid, ascorbic acid and o-nitrophenol. The PEDOT-rGO-Fe2O3-PPO biosensor was stable for at least 75 days when stored in a buffer at about 4°C. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain.

    Science.gov (United States)

    Caspers, Michael; Czogalla, Katrin J; Liphardt, Kerstin; Müller, Jens; Westhofen, Philipp; Watzka, Matthias; Oldenburg, Johannes

    2015-05-01

    VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways. Copyright © 2015. Published by Elsevier Ltd.

  17. The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

    Directory of Open Access Journals (Sweden)

    Colasanti Joseph

    2006-06-01

    Full Text Available Abstract Background The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD, which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. Results Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. Conclusion Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein

  18. Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

    Science.gov (United States)

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

  19. Structure-function analysis of human TYW2 enzyme required for the biosynthesis of a highly modified Wybutosine (yW base in phenylalanine-tRNA.

    Directory of Open Access Journals (Sweden)

    Virginia Rodriguez

    Full Text Available Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNA(Phe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2 is active in yeast and can synthesize the yW of yeast tRNA(Phe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet, and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNA(Phe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.

  20. Fabrication of highly electro catalytic active layer of multi walled carbon nanotube/enzyme for Pt-free dye sensitized solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Arbab, Alvira Ayoub, E-mail: alvira_arbab@yahoo.com [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Sun, Kyung Chul, E-mail: hytec@hanyang.ac.kr [Department of Fuel cells and hydrogen technology, Hanyang University, Seoul 133-791 (Korea, Republic of); Sahito, Iftikhar Ali, E-mail: iftikhar.sahito@faculty.muet.edu.pk [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Qadir, Muhammad Bilal, E-mail: bilal_ntu81@hotmail.com [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Jeong, Sung Hoon, E-mail: shjeong@hanyang.ac.kr [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of)

    2015-09-15

    Graphical abstract: - Highlights: • We prepared three different types of enzyme dispersed multiwall carbon nanotube (E-MWCNT) layer for application in Pt-free dye sensitized solar cell (DSSCs). • E-MWCNT catalysts exhibited an extremely good electro-catalytic activity (ECA), compared with the conventional catalyst, when synthesized with lipase enzyme. • E-MWCNT as counter electrode exhibits a high power conversion efficiency (PCE) of 7.5%, which can be compared to 8% efficiency of Pt catalyst. - Abstract: Highly dispersed conductive suspensions of multi walled carbon nanotubes (MWCNT) can have intrinsic electrical and electrochemical characteristics, which make them useful candidate for platinum (Pt)-free, dye sensitized solar cells (DSSCs). High energy conversion efficiency of 7.52% is demonstrated in DSSCs, based on enzyme dispersed MWCNT (E-MWCNT) layer deposited on fluorine doped tin oxide (FTO) glass. The E-MWCNT layer shows a pivotal role as platform to reduce large amount of iodide species via electro catalytically active layer, fabricated by facile tape casting under air drying technique. The E-MWCNT layer with large surface area, high mechanical adhesion, and good interconnectivity is derived from an appropriate enzyme dispersion, which provides not only enhanced interaction sites for the electrolyte/counter electrode interface but also improved electron transport mechanism. The surface morphology and structural characterization were investigated using field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), Raman spectroscopy and electronic microscopy techniques. Electro catalytic activity (ECA) and electrochemical properties of E-MWCNT counter electrode (CE) were investigated using cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements. The high power conversion efficiency (PCE) of E-MWCNT CE is associated with the low charge transfer

  1. Fabrication of highly electro catalytic active layer of multi walled carbon nanotube/enzyme for Pt-free dye sensitized solar cells

    International Nuclear Information System (INIS)

    Arbab, Alvira Ayoub; Sun, Kyung Chul; Sahito, Iftikhar Ali; Qadir, Muhammad Bilal; Jeong, Sung Hoon

    2015-01-01

    Graphical abstract: - Highlights: • We prepared three different types of enzyme dispersed multiwall carbon nanotube (E-MWCNT) layer for application in Pt-free dye sensitized solar cell (DSSCs). • E-MWCNT catalysts exhibited an extremely good electro-catalytic activity (ECA), compared with the conventional catalyst, when synthesized with lipase enzyme. • E-MWCNT as counter electrode exhibits a high power conversion efficiency (PCE) of 7.5%, which can be compared to 8% efficiency of Pt catalyst. - Abstract: Highly dispersed conductive suspensions of multi walled carbon nanotubes (MWCNT) can have intrinsic electrical and electrochemical characteristics, which make them useful candidate for platinum (Pt)-free, dye sensitized solar cells (DSSCs). High energy conversion efficiency of 7.52% is demonstrated in DSSCs, based on enzyme dispersed MWCNT (E-MWCNT) layer deposited on fluorine doped tin oxide (FTO) glass. The E-MWCNT layer shows a pivotal role as platform to reduce large amount of iodide species via electro catalytically active layer, fabricated by facile tape casting under air drying technique. The E-MWCNT layer with large surface area, high mechanical adhesion, and good interconnectivity is derived from an appropriate enzyme dispersion, which provides not only enhanced interaction sites for the electrolyte/counter electrode interface but also improved electron transport mechanism. The surface morphology and structural characterization were investigated using field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), Raman spectroscopy and electronic microscopy techniques. Electro catalytic activity (ECA) and electrochemical properties of E-MWCNT counter electrode (CE) were investigated using cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements. The high power conversion efficiency (PCE) of E-MWCNT CE is associated with the low charge transfer

  2. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  3. Method of preparing uridine-diphospho-/sup 14/C-D-glucose of high molar activity and radiochemical purity by enzyme synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, V; Biely, P; Koelbl, J

    1980-06-15

    A new method is described of enzyme synthesis of uridine-diphospho-/sup 14/C-D-glucose of high molar activity. After conversion of the initial /sup 14/C-D-glucose, the accompanying sugar phosphates and nucleoside-5'-phosphates are selectively hydrolyzed by the action of alkaline phosphatase in the presence of phenolphthalein as an indicator. Uridine-diphospho-/sup 14/C-D-glucose is separated by paper chromatography from the reaction mixture thus modified. Synthesis yields range within 75% and 85% relative to the starting /sup 14/C-D-glucose.

  4. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

    OpenAIRE

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-01-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstr...

  5. Ex Situ gene conservation in high elevation white pine species in the United States-a beginning

    Science.gov (United States)

    Richard A. Sniezko; Anna Schoettle; Joan Dunlap; Detlev Vogler; David Conklin; Andrew Bower; Chris Jensen; Rob Mangold; Doug Daoust; Gary Man

    2011-01-01

    The eight white pine species native to the western United States face an array of biotic and abiotic challenges that impact the viability of populations or the species themselves. Well-established programs are already in place to conserve and restore Pinus monticola Dougl. ex D. Don and P. lambertiana Dougl. throughout significant portions of their geographic ranges....

  6. A Highly Conserved GEQYQQLR Epitope Has Been Identified in the Nucleoprotein of Ebola Virus by Using an In Silico Approach

    Directory of Open Access Journals (Sweden)

    Mohammad Tuhin Ali

    2015-01-01

    Full Text Available Ebola virus (EBOV is a deadly virus that has caused several fatal outbreaks. Recently it caused another outbreak and resulted in thousands afflicted cases. Effective and approved vaccine or therapeutic treatment against this virus is still absent. In this study, we aimed to predict B-cell epitopes from several EBOV encoded proteins which may aid in developing new antibody-based therapeutics or viral antigen detection method against this virus. Multiple sequence alignment (MSA was performed for the identification of conserved region among glycoprotein (GP, nucleoprotein (NP, and viral structural proteins (VP40, VP35, and VP24 of EBOV. Next, different consensus immunogenic and conserved sites were predicted from the conserved region(s using various computational tools which are available in Immune Epitope Database (IEDB. Among GP, NP, VP40, VP35, and VP30 protein, only NP gave a 100% conserved GEQYQQLR B-cell epitope that fulfills the ideal features of an effective B-cell epitope and could lead a way in the milieu of Ebola treatment. However, successful in vivo and in vitro studies are prerequisite to determine the actual potency of our predicted epitope and establishing it as a preventing medication against all the fatal strains of EBOV.

  7. "Toward High School Biology": Helping Middle School Students Understand Chemical Reactions and Conservation of Mass in Nonliving and Living Systems

    Science.gov (United States)

    Herrmann-Abell, Cari F.; Koppal, Mary; Roseman, Jo Ellen

    2016-01-01

    Modern biology has become increasingly molecular in nature, requiring students to understand basic chemical concepts. Studies show, however, that many students fail to grasp ideas about atom rearrangement and conservation during chemical reactions or the application of these ideas to biological systems. To help provide students with a better…

  8. The expression and activity of antioxidant enzymes in the liver of rats exposed to high-fructose diet in the period from weaning to adulthood.

    Science.gov (United States)

    Glban, Alhadi M; Vasiljević, Ana; Veličković, Nataša; Nikolić-Kokić, Aleksandra; Blagojević, Duško; Matić, Gordana; Nestorov, Jelena

    2015-08-30

    Increased fructose consumption correlates with rising prevalence of various metabolic disorders, some of which were linked to oxidative stress. The relationship between fructose consumption and oxidative stress is complex and effects of a fructose-rich diet on the young population have not been fully elucidated. The aim of this study was to investigate whether high-fructose diet applied in the period from weaning to adulthood induces oxidative stress in the liver, thus contributing to induction or aggravation of metabolic disturbances in later adulthood. To that end we examined the effects of high-fructose diet on expression and activity of antioxidant enzymes, markers of lipid peroxidation and protein damage in the liver as the main fructose metabolizing tissue. High-fructose diet increased only SOD2 (mitochondrial manganese superoxide dismutase) activity, with no effect on other antioxidant enzymes, lipid peroxidation or accumulation of damaged proteins in the liver. The results show that fructose-induced metabolic disturbances could not be attributed to oxidative stress, at least not at young age. The absence of oxidative stress in the liver observed herein implies that young organisms are capable of maintaining redox homeostasis when challenged by fructose-derived energy overload. © 2014 Society of Chemical Industry.

  9. Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

    International Nuclear Information System (INIS)

    Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan; Park, Jong-Tae; Woo, Eui-Jeon; Kim, Myo-Jeong; Lee, Byong-Hoon; Park, Kwan-Hwa

    2009-01-01

    A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both α-(1,6)- and α-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with 14 C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-α-(1,6)-β-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

  10. Inhibitory control and visuo-spatial reversibility in Piaget’s seminal number conservation task: A high-density ERP study.

    Directory of Open Access Journals (Sweden)

    Gregoire eBorst

    2013-12-01

    Full Text Available The present high-density ERP study on 13 adults aimed to determine whether number conservation relies on the ability to inhibit the overlearned length-equals-number strategy and then imagine the shortening of the row that was lengthened. Participants performed the number-conservation task and, after the EEG session, the mental imagery task. In the number-conservation task, first two rows with the same number of tokens and the same length were presented on a computer screen (COV condition and then, the tokens in one of the two rows were spread apart (INT condition. Participants were instructed to determine whether the two rows had an identical number of tokens. In the mental imagery task, two rows with different lengths but the same number of tokens were presented and participants were instructed to imagine the tokens in the longer row aligning with the tokens in the shorter row. In the number-conservation task, we found that the amplitudes of the centro-parietal N2 and fronto-central P3 were higher in the INT than in the COV conditions. In addition, the differences in response times between the two conditions were correlated with the differences in the amplitudes of the fronto-central P3. In light of previous results reported on the number-conservation task in adults, the present results suggest that inhibition might be necessary to succeed the number-conservation task in adults even when the transformation of the length of one of the row is displayed. Finally, we also reported correlations between the speed at which participants could imagine the shortening of one of the row in the mental imagery task, the speed at which participants could determine that the two rows had the same number of tokens after the tokens in one of the row were spread apart and the latency of the late positive parietal component in the number-conservation task. Therefore, performing the number-conservation task might involve mental transformation processes in adults.

  11. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

  12. Strategies for enzyme saving during saccharification of pretreated lignocellulo-starch biomass: effect of enzyme dosage and detoxification chemicals

    Directory of Open Access Journals (Sweden)

    M.G. Mithra

    2017-08-01

    Full Text Available Two strategies leading to enzyme saving during saccharification of pretreated lignocellulo-starch biomass (LCSB was investigated which included reducing enzyme dosage by varying their levels in enzyme cocktails and enhancing the fermentable sugar yield in enzyme-reduced systems using detoxification chemicals. Time course release of reducing sugars (RS during 24–120 h was significantly higher when an enzyme cocktail containing full dose of cellulase (16 FPU/g cellulose along with half dose each of xylanase (1.5 mg protein/g hemicelluloses and Stargen (12.5 μl/g biomass was used to saccharify conventional dilute sulphuric acid (DSA pretreated biomass compared to a parallel system where only one-fourth the dose of the latter two enzymes was used. The reduction in RS content in the 120 h saccharified mash to the extent of 3–4 g/L compared to the system saccharified with full complement of the three enzymes could be overcome considerably by supplementing the system (half dose of two enzymes with detoxification chemical mix incorporating Tween 20, PEG 4000 and sodium borohydride. Microwave (MW-assisted DSA pretreated biomass on saccharification with enzyme cocktail having full dose of cellulase and half dose of Stargen along with detoxification chemicals gave significantly higher RS yield than DSA pretreated system saccharified using three enzymes. The study showed that xylanase could be eliminated during saccharification of MW-assisted DSA pretreated biomass without affecting RS yield when detoxification chemicals were also supplemented. The Saccharification Efficiency and Overall Conversion Efficiency were also high for the MW-assisted DSA pretreated biomass. Since whole slurry saccharifcation of pretreated biomass is essential to conserve fermentable sugars in LCSB saccharification, detoxification of soluble inhibitors is equally important as channelling out of insoluble lignin remaining in the residue. As one of the major factors contributing

  13. Toward mechanistic classification of enzyme functions.

    Science.gov (United States)

    Almonacid, Daniel E; Babbitt, Patricia C

    2011-06-01

    Classification of enzyme function should be quantitative, computationally accessible, and informed by sequences and structures to enable use of genomic information for functional inference and other applications. Large-scale studies have established that divergently evolved enzymes share conserved elements of structure and common mechanistic steps and that convergently evolved enzymes often converge to similar mechanisms too, suggesting that reaction mechanisms could be used to develop finer-grained functional descriptions than provided by the Enzyme Commission (EC) system currently in use. Here we describe how evolution informs these structure-function mappings and review the databases that store mechanisms of enzyme reactions along with recent developments to measure ligand and mechanistic similarities. Together, these provide a foundation for new classifications of enzyme function. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    Directory of Open Access Journals (Sweden)

    Raja Elina Raja Aziddin

    2011-08-01

    Full Text Available The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE on human recombinant cytochrome P450 (CYP enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC50 values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC50 of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC50 of CYP2C19 could not be determined, however, because inhibition was < 50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6, ketoconazole (CYP3A4, tranylcypromine (CYP2C19 and furafylline (CYP1A2 were used as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

  15. Process for preparing multilayer enzyme coating on a fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  16. Conservation endocrinology

    Science.gov (United States)

    McCormick, Stephen; Romero, L. Michael

    2017-01-01

    Endocrinologists can make significant contributions to conservation biology by helping to understand the mechanisms by which organisms cope with changing environments. Field endocrine techniques have advanced rapidly in recent years and can provide substantial information on the growth, stress, and reproductive status of individual animals, thereby providing insight into current and future responses of populations to changes in the environment. Environmental stressors and reproductive status can be detected nonlethally by measuring a number of endocrine-related endpoints, including steroids in plasma, living and nonliving tissue, urine, and feces. Information on the environmental or endocrine requirements of individual species for normal growth, development, and reproduction will provide critical information for species and ecosystem conservation. For many taxa, basic information on endocrinology is lacking, and advances in conservation endocrinology will require approaches that are both “basic” and “applied” and include integration of laboratory and field approaches.

  17. Determination of soluble dietary fibre content of Okara treated with high hydrostatic pressure and enzymes: a comparative evaluation of two methods (AOAC and HPLC-ELSD).

    Science.gov (United States)

    Pérez-López, E; Mateos-Aparicio, I; Rupérez, P

    2017-04-01

    Okara is a promising by-product from soybean and a rich source of dietary fibre, chiefly insoluble (IDF). To increase its solubility and functionality, a treatment with high hydrostatic pressure (HHP) assisted by food grade enzymes - Ultraflo ® L or Viscozyme ® L- has been performed. To monitor the effectiveness, an analysis of dietary fibre, mainly the soluble fraction (SDF), was accomplished by the AOAC enzymatic-gravimetric method with dialysis followed by both, spectrophotometric methods and High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD) analysis of the soluble fraction. A significant increase in SDF (≈1.5-times) to the expense of a decrease in IDF was shown and chromatograms revealed two peaks of 95 and 22 kDa. Thus, treated Okara possessed a more balanced and convenient ratio of soluble to insoluble dietary fibre, which could have health benefits as prebiotic. Inbuilt interferences of the official AOAC's method for dietary fibre were confirmed and the direct HPLC-ELSD approach was about twice more sensitive than spectrophotometric methods. Consequently, the direct HPLC-ELSD analysis of the supernatant is proposed as a cheaper, faster and reliable method. Combined HHP-treatments plus specific enzymes represent a promising alternative for the valorisation and preservation of agrofood by-products.

  18. Stabilization of red fruit-based smoothies by high-pressure processing. Part A. Effects on microbial growth, enzyme activity, antioxidant capacity and physical stability.

    Science.gov (United States)

    Hurtado, Adriana; Guàrdia, Maria Dolors; Picouet, Pierre; Jofré, Anna; Ros, José María; Bañón, Sancho

    2017-02-01

    Non-thermal pasteurization by high-pressure processing (HPP) is increasingly replacing thermal processing (TP) to maintain the properties of fresh fruit products. However, most of the research on HPP-fruit products only partially addresses fruit-pressure interaction, which limits its practical interest. The objective of this study was to assess the use of a mild HPP treatment to stabilize red fruit-based smoothies (microbial, enzymatic, oxidative and physical stability). HPP (350 MPa/10 °C/5 min) was slightly less effective than TP (85 °C/7 min) in inactivating microbes (mesophilic and psychrophilic bacteria, coliforms, yeasts and moulds) in smoothies kept at 4 °C for up to 28 days. The main limitation of using HPP was its low efficacy in inactivating oxidative (polyphenol oxidase and peroxidase) and hydrolytic (pectin methyl esterase) enzymes. Data on antioxidant status, colour parameters, browning index, transmittance, turbidity and viscosity confirmed that the HPP-smoothies have a greater tendency towards oxidation and clarification, which might lead to undesirable sensory and nutritional changes (see Part B). The microbial quality of smoothies was adequately controlled by mild HPP treatment without affecting their physical-chemical characteristics; however, oxidative and hydrolytic enzymes are highly pressure-resistant, which suggests that additional strategies should be used to stabilize smoothies. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. Multidisciplinary approach for the esophageal carcinoma with intent to conserve the esophagus centering on high-dose radiotherapy and concurrent chemotherapy

    International Nuclear Information System (INIS)

    Murakami, Masao; Kuroda, Yasumasa; Okamoto, Yoshiaki

    1997-01-01

    Forty-seven patients with operable squamous cell carcinoma of the thoracic esophagus were treated by initial concurrent chemoradiotherapy (CDDP-5 FU-44 Gy) followed by definitive high-dose of radiotherapy (CRT group: 35 patients) or surgery (CRT-S group: 12 patients). Clinical CR rate showed 86% in CRT group; and pathological CR rate 18% in CRT-S group. The overall median survival was 45 months, survival at 1, 3, 5 years being 96%, 52%, 48%, respectively. No treatment-related mortality was observed. The rate of the 'esophagus conservation' was 66%. Our results demonstrated that the multidisciplinary approach with intent to conserve the esophagus centering on high-dose radiotherapy and concurrent chemotherapy provides a significant improvement of both survival and quality of life in patients with operable esophageal carcinoma. (author)

  20. High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes.

    Science.gov (United States)

    Yu, Dan; Hornung, Ellen; Iven, Tim; Feussner, Ivo

    2018-01-01

    Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient. For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae . To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi ( Ab WSD1) and Marinobacter aquaeolei ( Ma WS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei ( Ma FAR) with Ab WSD1 or Ma WS2 led to a high incorporation of C 18 substrates in wax esters. The Ma FAR/TM Mm AWAT2- Ab WSD1 combination resulted in the incorporation of more C 18:1 alcohol and C 18:0 acyl moieties into wax esters compared with Ma FAR/ Ab WSD1. The fusion protein of a WS from Simmondsia chinensis ( Sc WS) with MaFAR exhibited higher specificity toward C 20:1 substrates in preference to C 18:1 substrates. Expression of Ma FAR/ Ab WSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by Ma FAR/ Ab WSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina , lines expressing Ma FAR/ Sc WS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed -1 of wax esters, containing 27-34 mol% oleyl oleate. The

  1. Highly transparent poly(2-ethyl-2-oxazoline)-TiO2 nanocomposite coatings for the conservation of matte painted artworks

    OpenAIRE

    Colombo, A.; Gherardi, Francesca; Goidanich, S.; Delaney, J. K.; de la Rie, E. R.; Ubaldi, M. C.; Toniolo, L.; Simonutti, R.

    2015-01-01

    A nanocomposite coating based on TiO2 nanoparticles and poly(2-ethyl-2-oxazoline) is used as consolidant of matte painted surfaces (temperas, watercolors, modern paintings). The aim of this work is to provide advances in the conservation of these works of art, while preserving their optical appearance, in terms of colour and gloss. Fiber Optic Reflectance Spectroscopy (FORS) measurements of a painting-model (an acrylic black monochrome) treated with the nanocomposite coatings revealed that it...

  2. A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

    Directory of Open Access Journals (Sweden)

    Nele Ilmberger

    Full Text Available A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs, which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

  3. DNA analysis indicates that Asian elephants are native to Borneo and are therefore a high priority for conservation.

    Directory of Open Access Journals (Sweden)

    Prithiviraj Fernando

    2003-10-01

    Full Text Available The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th-18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation.

  4. Implantable enzyme amperometric biosensors.

    Science.gov (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Cold-Adapted Enzymes

    Science.gov (United States)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  6. Combined osmodehydration and high pressure processing on the enzyme stability and antioxidant capacity of a grapefruit jam

    Science.gov (United States)

    A combined osmodehydration process and high pressure treatment (OD-HHP) was developed for grapefruit jam preservation. The inactivation kinetics of pectinmethylesterase (PME) and peroxidase (POD) in the osmodehydrated (OD) jam treated by combined thermal (45-75°C) and high pressure (550–700 MPa) pro...

  7. The selective conservative management of small traumatic pneumothoraces following stab injuries is safe: experience from a high-volume trauma service in South Africa.

    Science.gov (United States)

    Kong, V Y; Oosthuizen, G V; Clarke, D L

    2015-02-01

    The selective conservative management of small pneumothoraces (PTXs) following stab injuries is controversial. We reviewed a cohort of patients managed conservatively in a high volume trauma service in South Africa. A retrospective review over a 2-year period identified 125 asymptomatic patients with small PTXs measuring chest radiograph who were managed conservatively. Of the 125 patients included in the study, 92% were male (115/125), and the median age for all patients was 21 years (19-24). Ninety-seven per cent (121/125) of the weapons involved were knives, and 3% (4/125) were screwdrivers. Sixty-one per cent of all injuries were on the left side. Eighty-two per cent (102/125) sustained a single stab, and 18% (23/125) had multiple stabs. Thirty-nine per cent (49/125) had a PTX <0.5 cm (Group A), 26% (32/125) were ≥ 0.5 to <1 cm (Group B), 19% (24/125) were ≥ 1 to <1.5 cm (Group C) and 15% (20/125) were ≥ 1.5 to <2 cm (Group D). Three per cent of all patients (4/125) eventually required ICDs (one in Group C, three in Group D). All four patients had ICDs in situ for 24 h. The remaining 97% (121/125) were all managed successfully by active clinical observation alone. There were no subsequent readmissions, morbidity or mortality as a direct result of our conservative approach. The selective conservative management of asymptomatic small PTXs from stab injuries is safe if undertaken in the appropriate setting.

  8. A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes

    International Nuclear Information System (INIS)

    Salvucci, M.E.; Crafts-Brandner, S.J.

    1991-01-01

    A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, [14C]sucrose phosphate, from unreacted uridine 5'-diphosphate-[14C]glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here the authors show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts

  9. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  10. [Conservation Units.

    Science.gov (United States)

    Texas Education Agency, Austin.

    Each of the six instructional units deals with one aspect of conservation: forests, water, rangeland, minerals (petroleum), and soil. The area of the elementary school curriculum with which each correlates is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the…

  11. Creative conservation

    NARCIS (Netherlands)

    Bentham, Roelof J.

    1968-01-01

    The increasing exploitation of our natural resources, the unlimited occupation of ever more new areas, and the intensification of land-use, make it necessary for us to expand the concept of conservation. But we also need to reconsider that concept itself. For the changing conditions in the

  12. Reshaping conservation

    DEFF Research Database (Denmark)

    Funder, Mikkel; Danielsen, Finn; Ngaga, Yonika

    2013-01-01

    members strengthen the monitoring practices to their advantage, and to some extent move them beyond the reach of government agencies and conservation and development practitioners. This has led to outcomes that are of greater social and strategic value to communities than the original 'planned' benefits...

  13. The effect of germination and metallic salts on the stability of enzymes of three high yielding varieties of maize (Zea mays L. in respect of Bangladesh

    Directory of Open Access Journals (Sweden)

    Rupa, A.Z.,

    2017-09-01

    Full Text Available The study was conducted with a view to determine the effect of germination and metallic salts on nutritional quality, enzymes activity and their stability of three high yielding varieties of maize (Zea mays L.. The protein content of BHM-3, BHM-5 and BHM-6 were increased 22.37%, 26.48%, and 20.34% respectively at 48 hours then decreased drastically from 72-96 hours of germination. Starch content was increased maximum 29.19% in BHM-6 at 0 hours (non-germinating seeds among the three varieties and then decreased gradually from 48-96 hours of germination. Total sugar and reducing sugar contents of BHM-3, BHM-5 and BHM-6 seeds were maximum at 96 hours than 24-72 hours of germination while BHM-3 showed boosting increase of total sugar (336.97% due to 96 hours of germination. BHM-5 showed a tremendous increase of α-amylase (189.83% and protease (144.44% activity whereas BHM-6 showed maximum invertase activity (175.27% at 48 hours then decreased gradually from 72-96 hours of germination. The activities of enzymes were increased in presence of metallic salts such as Ca2+, Mg2+, and Mn2+ while Fe2+, Zn2+ and Cu2+ inhibited the activities moderately.

  14. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    International Nuclear Information System (INIS)

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker

    2005-01-01

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K m ) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 μM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors

  15. Integrating conservation costs into sea level rise adaptive conservation prioritization

    Directory of Open Access Journals (Sweden)

    Mingjian Zhu

    2015-07-01

    Full Text Available Biodiversity conservation requires strategic investment as resources for conservation are often limited. As sea level rises, it is important and necessary to consider both sea level rise and costs in conservation decision making. In this study, we consider costs of conservation in an integrated modeling process that incorporates a geomorphological model (SLAMM, species habitat models, and conservation prioritization (Zonation to identify conservation priorities in the face of landscape dynamics due to sea level rise in the Matanzas River basin of northeast Florida. Compared to conservation priorities that do not consider land costs in the analysis process, conservation priorities that consider costs in the planning process change significantly. The comparison demonstrates that some areas with high conservation values might be identified as lower priorities when integrating economic costs in the planning process and some areas with low conservation values might be identified as high priorities when considering costs in the planning process. This research could help coastal resources managers make informed decisions about where and how to allocate conservation resources more wisely to facilitate biodiversity adaptation to sea level rise.

  16. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose...

  17. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

    International Nuclear Information System (INIS)

    Kwon, Y.K.; Hecht, N.B.

    1991-01-01

    The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

  18. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  19. The Highly Conserved COPII Coat Complex Sorts Cargo from the Endoplasmic Reticulum and Targets It to the Golgi

    OpenAIRE

    Lord, Christopher; Ferro-Novick, Susan; Miller, Elizabeth A.

    2013-01-01

    Protein egress from the endoplasmic reticulum (ER) is driven by a conserved cytoplasmic coat complex called the COPII coat. The COPII coat complex contains an inner shell (Sec23/Sec24) that sorts cargo into ER-derived vesicles and an outer cage (Sec13/Sec31) that leads to coat polymerization. Once released from the ER, vesicles must tether to and fuse with the target membrane to deliver their protein and lipid contents. This delivery step also depends on the COPII coat, with coat proteins bin...

  20. Research progress of nanoparticles as enzyme mimetics

    Science.gov (United States)

    Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun

    2011-10-01

    Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

  1. Growth and enzyme production during continuous cultures of a high amylase-producing variant of Aspergillus oryzae

    DEFF Research Database (Denmark)

    Zangirolami, Teresa; Carlsen, M.; Nielsen, J.

    2002-01-01

    Growth and product formation by a selected variant of Aspergillus oryzae showing high alpha-amylase production was studied in continuous cultivations carried out at six different specific growth rates, using glucose as the growth-limiting nutrient. The analysis of the steady-state data revealed...

  2. Conservation of Charge and Conservation of Current

    OpenAIRE

    Eisenberg, Bob

    2016-01-01

    Conservation of current and conservation of charge are nearly the same thing: when enough is known about charge movement, conservation of current can be derived from conservation of charge, in ideal dielectrics, for example. Conservation of current is enforced implicitly in ideal dielectrics by theories that conserve charge. But charge movement in real materials like semiconductors or ionic solutions is never ideal. We present an apparently universal derivation of conservation of current and ...

  3. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

    Directory of Open Access Journals (Sweden)

    Md. Tariqul Islam

    2015-01-01

    Full Text Available MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

  4. Relatively High Complication and Revision Rates of the Mayo® Metaphysical Conservative Femoral Stem in Young Patients.

    Science.gov (United States)

    Rutenberg, Tal Frenkel; Warshevski, Yaniv; Gold, Aviram; Shasha, Nadav; Snir, Nimrod; Chechik, Ofir; Dolkart, Oleg; Eilig, Dynai; Herman, Amir; Rath, Ehud; Kramer, Moti; Drexler, Michael

    2018-05-08

    The Mayo metaphysical conservative femoral stem (Zimmer, Warsaw, Indiana) is a wedge-shaped implant designed to transfer loads proximally, reduce femoral destruction, and enable the preservation of bone stock in the proximal femur. Thus, it is a potentially preferred prosthesis for active, non-elderly patients who may require additional future surgeries. This retrospective case study analyzed the outcomes of consecutive patients who underwent total hip replacements with this stem between May 2001 and February 2013. All patients underwent clinical assessment, radiological evaluation for the presence and development of radiolucent lines, and functional assessment (numerical analog scale, Harris hip score, and Short Form-12 questionnaire). Ninety-five hips (79 patients) were available for analysis. The patients' mean age was 43 years (range, 18-64 years), and the mean follow-up was 97 months (range, 26.9-166 months). The postoperative clinical assessments and functional assessments revealed significant improvements. Sixteen patients (20.3%) had 18 orthopedic complications, the most common of which were an intraoperative femoral fracture and implant dislocation requiring revision surgeries in 10 hips (10.5%). Radiological analysis revealed evidence of femoral remodeling in 64 (67.4%) implants, spot welds (neocortex) in 35 (36.8%), and osteolysis in 3 (3.2%). These results suggest that the conservative hip femoral implant has an unacceptable complication rate for non-elderly patients. [Orthopedics. 201x; xx(x):xx-xx.]. Copyright 2018, SLACK Incorporated.

  5. Serum, plasma, and dried blood spot high sensitivity C-reactive protein enzyme immunoassay for population research

    OpenAIRE

    Brindle, Eleanor; Fujita, Masako; Shofer, Jane; O’Connor, Kathleen A.

    2010-01-01

    C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus need for a robust high sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to...

  6. Inhibition of angiotensin-1-converting enzyme activity by two varieties of ginger (Zingiber officinale) in rats fed a high cholesterol diet.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Ademiluyi, Adedayo Oluwaseun; Oboh, Ganiyu

    2014-03-01

    Angiotensin-1-converting enzyme (ACE) inhibitors are widely used in the treatment of cardiovascular diseases. This study sought to investigate the inhibitory effect of two varieties of ginger (Zingiber officinale) commonly consumed in Nigeria on ACE activity in rats fed a high cholesterol diet. The inhibition of ACE activity of two varieties of ginger (Z. officinale) was investigated in a high cholesterol (2%) diet fed to rats for 3 days. Feeding high cholesterol diets to rats caused a significant (Pginger varieties. Rats that were fed 4% white ginger had the greatest inhibitory effect as compared with a control diet. Furthermore, there was a significant (Pginger (either 2% or 4%) caused a significant (Pginger had the greatest reduction as compared with control diet. In conclusion, both ginger varieties exhibited anti-hypercholesterolemic properties in a high cholesterol diet fed to rats. This activity of the gingers may be attributed to its ACE inhibitory activity. However, white ginger inhibited ACE better in a high cholesterol diet fed to rats than red ginger. Therefore, both gingers could serve as good functional foods/nutraceuticals in the management/treatment of hypertension and other cardiovascular diseases.

  7. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

    Directory of Open Access Journals (Sweden)

    Ukkonen Kaisa

    2011-12-01

    Full Text Available Abstract This report describes the combined use of an enzyme-based glucose release system (EnBase® and high-aeration shake flask (Ultra Yield Flask™. The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

  8. Diesel conservation: GSRTC'S experience

    Energy Technology Data Exchange (ETDEWEB)

    Ramesh Kumar, I V

    1980-01-01

    The Gujarat State Road Transport Corporation (GSRTC) in India has a fleet of about 6000 buses. The increasing cost of fuel and lubricants added to uncertainty in supplies, has necessitated the need for conserving High Speed Diesel Oil (HSD). GSRTC had achieved an overall average Kilometre Per Litre (kmpl) of 4.44 in the year 1976-1977 due to a variety of measures. In the year 1978-1979 the average kmpl was 4.52 and it is expected to be 4.60 for 1979-1980. The case study outlined describes the measures taken by GSRTC in conserving high speed diesel oil by various methods.

  9. Influence of high glycine diets on the activity of glycine-catabolizing enzymes and on glycine catabolism in rats

    International Nuclear Information System (INIS)

    Petzke, K.J.; Albrecht, V.; Przybilski, H.

    1986-01-01

    Male albino rats were adapted to isocaloric purified diets that differed mainly in their glycine and casein contents. Controls received a 30% casein diet. In experimental diets gelatin or gelatin hydrolysate was substituted for half of the 30% casein. An additional group was fed a glycine-supplemented diet, which corresponded in glycine level to the gelatin diet but in which the protein level was nearly the same as that of the casein control diet. Another group received a 15% casein diet. Rat liver glycine cleavage system, serine hydroxymethyltransferase and serine dehydratase activities were measured. 14 CO 2 production from the catabolism of 14 C-labeled glycine was measured in vivo and in vitro (from isolated hepatocytes). Serine dehydratase and glycine cleavage system activities were higher in animals fed 30% casein diets than in those fed 15% casein diets. Serine hydroxymethyltransferase activity of the cytosolic and mitochondrial fractions was highest when a high glycine diet (glycine administered as pure, protein bound in gelatin or peptide bound in gelatin hydrolysate) was fed. 14 CO 2 formation from [1- 14 C]- and [2- 14 C]glycine both in vivo and in isolated hepatocytes was higher when a high glycine diet was fed than when a casein diet was fed. These results suggest that glycine catabolism is dependent on and adaptable to the glycine content of the diet. Serine hydroxymethyltransferase appears to play a major role in the regulation of glycine degradation via serine and pyruvate

  10. Comparative interactomics: analysis of arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants.

    Science.gov (United States)

    Paul, Anna-Lisa; Liu, Li; McClung, Scott; Laughner, Beth; Chen, Sixue; Ferl, Robert J

    2009-04-01

    As a first step in the broad characterization of plant 14-3-3 multiprotein complexes in vivo, stringent and specific antibody affinity purification was used to capture 14-3-3s together with their interacting proteins from extracts of Arabidopsis cell suspension cultures. Approximately 120 proteins were identified as potential in vivo 14-3-3 interacting proteins by mass spectrometry of the recovered complexes. Comparison of the proteins in this data set with the 14-3-3 interacting proteins from a similar study in human embryonic kidney cell cultures revealed eight interacting proteins that likely represent reasonably abundant, fundamental 14-3-3 interaction complexes that are highly conserved across all eukaryotes. The Arabidopsis 14-3-3 interaction data set was also compared to a yeast in vivo 14-3-3 interaction data set. Four 14-3-3 interacting proteins are conserved in yeast, humans, and Arabidopsis. Comparisons of the data sets based on biochemical function revealed many additional similarities in the human and Arabidopsis data sets that represent conserved functional interactions, while also leaving many proteins uniquely identified in either Arabidopsis or human cells. In particular, the Arabidopsis interaction data set is enriched for proteins involved in metabolism.

  11. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  12. What is a conservation actor?

    Directory of Open Access Journals (Sweden)

    Paul Jepson

    2011-01-01

    Full Text Available As a crisis-oriented discipline, conservation biology needs actions to understand the state of nature and thwart declines in biodiversity. Actors-traditionally individuals, institutions, and collectives-have been central to delivering such goals in practice. However, the definition of actors within the discipline has been narrow and their role in influencing conservation outcomes inadequately conceptualised. In this paper, we examine the question ′What is a conservation actor?′ Who or what creates the capacity to influence conservation values and actions? Drawing from theoretical developments in Actor-Network Theory and collective governance, we argue that the concept of an actor in conservation biology should be broadened to include non-humans, such as species and devices, because they have the agency and ability to influence project goals and outcomes. We illustrate this through four examples: the Asian elephant, International Union for Conservation of Nature red lists, the High Conservation Value approach, and an Integrated Conservation and Development Project. We argue that a broader conceptualisation of actors in conservation biology will produce new forms of understanding that could open up new areas of conservation research, enhance practice and draw attention to spheres of conservation activity that might require stronger oversight and governance.

  13. Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

    Science.gov (United States)

    Loureiro, Bárbara; Ereno, Ronaldo L; Favoreto, Mauricio G; Barros, Ciro M

    2016-07-15

    Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice

    Directory of Open Access Journals (Sweden)

    Margarita Vida

    2015-07-01

    Full Text Available Interleukin-6 (IL-6 has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD in wild-type (WT and IL-6-deficient (IL-6−/− mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6. Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1 and signal transducer and activator of transcription-3 (STAT3, increased AMP kinase phosphorylation (p-AMPK, and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS and stearoyl-CoA desaturase 1 (SCD1. The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β, FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis.

  15. Determination of 5 '-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Nielsen, Jens

    1999-01-01

    We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length...... (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide...

  16. UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes

    International Nuclear Information System (INIS)

    Antonenkov, Vasily D.; Ohlmeier, Steffen; Sormunen, Raija T.; Hiltunen, J. Kalervo

    2007-01-01

    Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism

  17. Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

    Science.gov (United States)

    Ghoshroy, Sohini; Robertson, Deborah L

    2015-01-01

    Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

  18. A high-order finite-volume method for hyperbolic conservation laws on locally-refined grids

    Energy Technology Data Exchange (ETDEWEB)

    McCorquodale, Peter; Colella, Phillip

    2011-01-28

    We present a fourth-order accurate finite-volume method for solving time-dependent hyperbolic systems of conservation laws on Cartesian grids with multiple levels of refinement. The underlying method is a generalization of that in [5] to nonlinear systems, and is based on using fourth-order accurate quadratures for computing fluxes on faces, combined with fourth-order accurate Runge?Kutta discretization in time. To interpolate boundary conditions at refinement boundaries, we interpolate in time in a manner consistent with the individual stages of the Runge-Kutta method, and interpolate in space by solving a least-squares problem over a neighborhood of each target cell for the coefficients of a cubic polynomial. The method also uses a variation on the extremum-preserving limiter in [8], as well as slope flattening and a fourth-order accurate artificial viscosity for strong shocks. We show that the resulting method is fourth-order accurate for smooth solutions, and is robust in the presence of complex combinations of shocks and smooth flows.

  19. Toward High School Biology: Helping Middle School Students Understand Chemical Reactions and Conservation of Mass in Nonliving and Living Systems

    Science.gov (United States)

    Herrmann-Abell, Cari F.; Koppal, Mary; Roseman, Jo Ellen

    2016-01-01

    Modern biology has become increasingly molecular in nature, requiring students to understand basic chemical concepts. Studies show, however, that many students fail to grasp ideas about atom rearrangement and conservation during chemical reactions or the application of these ideas to biological systems. To help provide students with a better foundation, we used research-based design principles and collaborated in the development of a curricular intervention that applies chemistry ideas to living and nonliving contexts. Six eighth grade teachers and their students participated in a test of the unit during the Spring of 2013. Two of the teachers had used an earlier version of the unit the previous spring. The other four teachers were randomly assigned either to implement the unit or to continue teaching the same content using existing materials. Pre- and posttests were administered, and the data were analyzed using Rasch modeling and hierarchical linear modeling. The results showed that, when controlling for pretest score, gender, language, and ethnicity, students who used the curricular intervention performed better on the posttest than the students using existing materials. Additionally, students who participated in the intervention held fewer misconceptions. These results demonstrate the unit’s promise in improving students’ understanding of the targeted ideas. PMID:27909024

  20. Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence.

    Science.gov (United States)

    Benko, Sabina; Fantes, Judy A; Amiel, Jeanne; Kleinjan, Dirk-Jan; Thomas, Sophie; Ramsay, Jacqueline; Jamshidi, Negar; Essafi, Abdelkader; Heaney, Simon; Gordon, Christopher T; McBride, David; Golzio, Christelle; Fisher, Malcolm; Perry, Paul; Abadie, Véronique; Ayuso, Carmen; Holder-Espinasse, Muriel; Kilpatrick, Nicky; Lees, Melissa M; Picard, Arnaud; Temple, I Karen; Thomas, Paul; Vazquez, Marie-Paule; Vekemans, Michel; Roest Crollius, Hugues; Hastie, Nicholas D; Munnich, Arnold; Etchevers, Heather C; Pelet, Anna; Farlie, Peter G; Fitzpatrick, David R; Lyonnet, Stanislas

    2009-03-01

    Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

  1. High spatial resolution mapping of the Cerrado's land cover and land use types in the priority area for conservation Chapada da Contagem, Brazil.

    Science.gov (United States)

    Ribeiro, F.; Roberts, D. A.; Davis, F. W.; Antunes Daldegan, G.; Nackoney, J.; Hess, L. L.

    2016-12-01

    The Brazilian savanna, Cerrado, is the second largest biome over South America and the most floristically diverse savanna in the world. This biome is considered a conservation hotspot in respect to its biodiversity importance and rapid transformation of its landscape. The Cerrado's natural vegetation has been severely transformed by agriculture and pasture activities. Currently it is the main agricultural frontier in Brazil and one of the most threatened Brazilian biomes. This scenario results in environmental impacts such as ecosystems fragmentation as well as losses in connectivity, biodiversity and gene flow, changes in the microclimate and energy, carbon and nutrients cycles, among others. The Priority Areas for Conservation is a governmental program from Brazil that identifies areas with high conservation priority. One of this program's recommendation is a natural vegetation map including their major ecosystem classes. This study aims to generate more precise information for the Cerrado's vegetation. The main objective of this study is to identify which ecosystems are being prioritized and/or threatened by land use, refining information for further protection. In order to test methods, the priority area for conservation Chapada da Contagem was selected as the study site. This area is ranked as "extremely high priority" by the government and is located in the Federal District and Goias State, Brazil. Satellites with finer spatial resolution may improve the classification of the Cerrado's vegetation. Remote sensing methods and two criteria were tested using RapidEye 3A imagery (5m spatial resolution) collected in 2014 in order to classify the Cerrado's major land cover types of this area, as well as its land use. One criterion considers the Cerrado's major terrestrial ecosystems, which are divided into forest, savanna and grassland. The other involves scaling it down to the major physiognomic groups of each ecosystem. Other sources of environmental dataset such

  2. Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level

    Directory of Open Access Journals (Sweden)

    Henrissat Bernard

    2011-01-01

    Full Text Available Abstract Background Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses. Results Carbohydrate Active enzyme (CAZy annotation of the R. oryzae identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs and a high number of glycosyl transferases (GTs and carbohydrate esterases (CEs. A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars, chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of R. oryzae to its environment. Conclusions CAZy analyses of the genome of the zygomycete fungus R. oryzae and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.

  3. Inkjet-assisted layer-by-layer printing of quantum dot/enzyme microarrays for highly sensitive detection of organophosphorous pesticides.

    Science.gov (United States)

    Luan, Enxiao; Zheng, Zhaozhu; Li, Xinyu; Gu, Hongxi; Liu, Shaoqin

    2016-04-15

    We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time-saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5-100 μg L(-1) under the optimized conditions with the limit of detection (LOD) of 10 μg L(-1). The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS). Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Impact of angiotensin-converting enzyme gene polymorphism on neurohormonal responses to high- versus low-dose enalapril in advanced heart failure.

    Science.gov (United States)

    Tang, W H Wilson; Vagelos, Randall H; Yee, Yin-Gail; Fowler, Michael B

    2004-11-01

    The impact of angiotensin-converting enzyme (ACE) gene polymorphism on neurohormonal dose response to ACE inhibitor therapy is unclear. ACE Insertion (I) or Deletion (D) genotype was determined in 74 patients with chronic heart failure who were randomly assigned to receive either high-dose or low-dose enalapril over a period of 6 months. Monthly pre-enalapril and post-enalapril neurohormone levels (serum ACE activity (sACE), plasma angiotensin II (A-II), plasma renin activity (PRA), and serum aldosterone (ALDO) were compared between genotype subgroups and between patients who received high- or low-dose enalapril within each genotype subgroup. At baseline, predose/postdose sACE and postdose PRA were significantly higher in the DD genotype. At 6-month follow-up, postdose sACE was reduced in a dose-dependent fashion in all three genotypes (P sACE were consistently higher in the DD genotype when compared with ID or II subgroups. Despite a dose-dependent suppression of sACE, there were no observed statistically significant differences in ALDO and A-II suppression or escape with escalating doses of enalapril within each subgroup.

  5. Conservation of Mangifera sylvatica

    DEFF Research Database (Denmark)

    Akhter, Sayma

    and conservation of these valuable species. The present study considers an underutilised and threatened species of Bangladesh, namely wild mango (Mangifera sylvatica Roxb.). Although this wild mango is one of the genetically closest species to the common mango (Mangifera indica L.) research is very limited...... and mostly focused on wood quality and phylogenetic relationships. Therefore, this study investigated the conservation potential of wild mango considering its contribution for food, nutrition and livelihoods. To do so, an assessment was made of the current and future distribution of the species, which...... explored. The study conveyed five key messages: 1. Wild mango may become extinct under future climate change scenarios so it is high time to start thinking about conservation initiatives. 2. Wild mango is a small sized mango with a large kernel in relation to other Mangifera species which provides...

  6. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  7. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  8. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

    Directory of Open Access Journals (Sweden)

    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  9. Toward High School Biology: Helping Middle School Students Understand Chemical Reactions and Conservation of Mass in Nonliving and Living Systems.

    Science.gov (United States)

    Herrmann-Abell, Cari F; Koppal, Mary; Roseman, Jo Ellen

    2016-01-01

    Modern biology has become increasingly molecular in nature, requiring students to understand basic chemical concepts. Studies show, however, that many students fail to grasp ideas about atom rearrangement and conservation during chemical reactions or the application of these ideas to biological systems. To help provide students with a better foundation, we used research-based design principles and collaborated in the development of a curricular intervention that applies chemistry ideas to living and nonliving contexts. Six eighth grade teachers and their students participated in a test of the unit during the Spring of 2013. Two of the teachers had used an earlier version of the unit the previous spring. The other four teachers were randomly assigned either to implement the unit or to continue teaching the same content using existing materials. Pre- and posttests were administered, and the data were analyzed using Rasch modeling and hierarchical linear modeling. The results showed that, when controlling for pretest score, gender, language, and ethnicity, students who used the curricular intervention performed better on the posttest than the students using existing materials. Additionally, students who participated in the intervention held fewer misconceptions. These results demonstrate the unit's promise in improving students' understanding of the targeted ideas. © 2016 C. F. Herrmann-Abell et al. CBE—Life Sciences Education © 2016 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  10. An attempt to conserve whatnot somniferum (L.) dual - a highly essential medicinal plant, through in vitro callus culture

    International Nuclear Information System (INIS)

    Rout, J.R.; Sahoo, S.

    2011-01-01

    A simple effective protocol was developed for conservation and plant propagation through callus cultures of Withania somnifera (Ashwagandha). Seed germination percentage reached a maximum value of 64.3% on half MS + GA3 0.25 mg/l at third week of culture. Three different basal media compared for seed germination, MS was most effective. Out of 25 combinations of growth regulators evaluated, MS + 1.0 mg/l BA + 1.0 mg/l 2, 4-D found to be best for callus induction and proliferation regardless to explants. Among the four different explants tested, In vivo leaf explant was found most suitable for callus induction, proliferation and fresh weight gain. The highest callus induction frequency percentage 86.4% was recorded with In vivo leaf explant whereas, 43.4% in In vitro leaf explant at day 30 on MS augmented with 1.0 mg/l BA + 1.0 mg/l 2,4-D. Among different growth regulator combinations tested in augmentation with MS for shoot initiation and elongation, 2.0 mg/l BA + 1.0 mg/l NAA was the best eliciting a maximum of 82.3% shoot induction with highest shoot number 4.8 shoots/callus. The original callus was sub-cultured 2 times on fresh shoot multiplication medium after each harvest of the shoots. Of three different auxins tested for In vitro rooting, IBA was most effective compared to IPA and NAA. Half-strength MS medium containing IBA at an optimum concentration of 2.0 mg/l induced rooting in 83.1% of the In vitro derived shoots. The rooted plantlets were acclimated and eventually established in soil. (author)

  11. A Key Enzyme of the NAD+ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures.

    Science.gov (United States)

    Taniguchi, Hironori; Sungwallek, Sathidaphorn; Chotchuang, Phatcharin; Okano, Kenji; Honda, Kohsuke

    2017-09-01

    NAD (NAD + ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD + from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD + de novo synthesis pathway. Herein, we report the importance of the NAD + salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD + salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide ( K m of 17 μM, k cat of 50 s -1 , k cat / K m of 3.0 × 10 3 s -1 · mM -1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD + salvage pathway in T. thermophilus Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD + and NADH. IMPORTANCE NAD + and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD + decomposition is, in general, more rapid. This study emphasizes that NAD + instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures. Copyright © 2017 American Society for Microbiology.

  12. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  13. Biocatalytic material comprising multilayer enzyme coated fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  14. Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities.

    Science.gov (United States)

    Vouille, V; Amiche, M; Nicolas, P

    1997-09-01

    We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon 1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys-Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3'-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous 'secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.

  15. Comprehensive Non-Destructive Conservation Documentation of Lunar Samples Using High-Resolution Image-Based 3D Reconstructions and X-Ray CT Data

    Science.gov (United States)

    Blumenfeld, E. H.; Evans, C. A.; Oshel, E. R.; Liddle, D. A.; Beaulieu, K.; Zeigler, R. A.; Hanna, R. D.; Ketcham, R. A.

    2015-01-01

    Established contemporary conservation methods within the fields of Natural and Cultural Heritage encourage an interdisciplinary approach to preservation of heritage material (both tangible and intangible) that holds "Outstanding Universal Value" for our global community. NASA's lunar samples were acquired from the moon for the primary purpose of intensive scientific investigation. These samples, however, also invoke cultural significance, as evidenced by the millions of people per year that visit lunar displays in museums and heritage centers around the world. Being both scientifically and culturally significant, the lunar samples require a unique conservation approach. Government mandate dictates that NASA's Astromaterials Acquisition and Curation Office develop and maintain protocols for "documentation, preservation, preparation and distribution of samples for research, education and public outreach" for both current and future collections of astromaterials. Documentation, considered the first stage within the conservation methodology, has evolved many new techniques since curation protocols for the lunar samples were first implemented, and the development of new documentation strategies for current and future astromaterials is beneficial to keeping curation protocols up to date. We have developed and tested a comprehensive non-destructive documentation technique using high-resolution image-based 3D reconstruction and X-ray CT (XCT) data in order to create interactive 3D models of lunar samples that would ultimately be served to both researchers and the public. These data enhance preliminary scientific investigations including targeted sample requests, and also provide a new visual platform for the public to experience and interact with the lunar samples. We intend to serve these data as they are acquired on NASA's Astromaterials Acquisistion and Curation website at http://curator.jsc.nasa.gov/. Providing 3D interior and exterior documentation of astromaterial

  16. Mammals of the high altitudes of western Arunachal Pradesh, eastern Himalaya: an assessment of threats and conservation needs

    NARCIS (Netherlands)

    Mishra, C.; Madhusudan, M.D.; Datta, A.

    2006-01-01

    he high altitudes of Arunachal Pradesh, India, located in the Eastern Himalaya biodiversity hotspot, remain zoologically unexplored and unprotected. We report results of recent mammal surveys in the high altitude habitats of western Arunachal Pradesh. A total of 35 mammal species (including 12

  17. Plant Products for Pharmacology: Application of Enzymes in Their Transformations

    Directory of Open Access Journals (Sweden)

    Marie Zarevúcka

    2008-12-01

    Full Text Available Different plant products have been subjected to detailed investigations due to their increasing importance for improving human health. Plants are sources of many groups of natural products, of which large number of new compounds has already displayed their high impact in human medicine. This review deals with the natural products which may be found dissolved in lipid phase (phytosterols, vitamins etc.. Often subsequent convenient transformation of natural products may further improve the pharmacological properties of new potential medicaments based on natural products. To respect basic principles of sustainable and green procedures, enzymes are often employed as efficient natural catalysts in such plant product transformations. Transformations of lipids and other natural products under the conditions of enzyme catalysis show increasing importance in environmentally safe and sustainable production of pharmacologically important compounds. In this review, attention is focused on lipases, efficient and convenient biocatalysts for the enantio- and regioselective formation / hydrolysis of ester bond in a wide variety of both natural and unnatural substrates, including plant products, eg. plant oils and other natural lipid phase compounds. The application of enzymes for preparation of acylglycerols and transformation of other natural products provides big advantage in comparison with employing of conventional chemical methods: Increased selectivity, higher product purity and quality, energy conservation, elimination of heavy metal catalysts, and sustainability of the employed processes, which are catalyzed by enzymes. Two general procedures are used in the transformation of lipid-like natural products: (a Hydrolysis/alcoholysis of triacylglycerols and (b esterification of glycerol. The reactions can be performed under conventional conditions or in supercritical fluids/ionic liquids. Enzyme-catalyzed reactions in supercritical fluids combine the

  18. The relationship between the Resource Conservation and Recovery Act and the storage and disposal of spent nuclear fuel and high-level waste

    International Nuclear Information System (INIS)

    Gertz, C.P.; Cloke, P.L.

    1993-01-01

    This paper addresses the potential applicability of the requirements of the Resource Conservation and Recovery Act (RCRA) to the disposal of spent commercial nuclear fuel and of high-level (vitrified) radioactive waste. The Atomic Energy Act of 1954, as amended, and the associated regulations issued by the US NRC provides many requirements that apply to these waste forms and largely, if not entirely, pre-empts the applicability of RCRA. The RCRA would apply only to the non-radioactive components of these wastes, and then only in respect to hazardous components. In view of these restrictions it becomes important to evaluate whether any components of spent fuel or high-level waste are toxic, as defined by the RCRA regulations. Present indications are that they are not and, hence, the US DOE is proceeding on the basis that these wastes and others that may be generated in the future are non-hazardous in respect to RCRA definitions

  19. Inkjet-assisted layer-by-layer printing of quantum dot/enzyme microarrays for highly sensitive detection of organophosphorous pesticides

    Energy Technology Data Exchange (ETDEWEB)

    Luan, Enxiao; Zheng, Zhaozhu; Li, Xinyu; Gu, Hongxi [State Key Laboratory of Urban Water Resource and Environment, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080 (China); Micro- and Nanotechnology Research Center, Harbin Institute of Technology, Harbin 150080 (China); Liu, Shaoqin, E-mail: shaoqinliu@hit.edu.cn [Micro- and Nanotechnology Research Center, Harbin Institute of Technology, Harbin 150080 (China)

    2016-04-15

    We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time–saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5–100 μg L{sup −1} under the optimized conditions with the limit of detection (LOD) of 10 μg L{sup −1}. The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS). - Graphical abstract: A fluorimetric assay for high-throughput screening of organophosphorous pesticides was developed based on the CdTe QDs/AChE microarrays via inkjet-assisted LbL printing techniques. - Highlights: • The large scale microarrays of CdTe QDs and AChE were fabricated by facile inkjet-assisted LbL printing technique. • The QDs/AChE microscopic dot arrays could be used quantitatively and rapidly for the sensitively visual detection of OPs. • A detection limit of 10 μg L{sup −1} was achieved, much lower than levels specified by standard tests and other colorimetric detection methods. • The low cost, short processing time, sufficient sensitivity, good stability and ease of use make it for a facile platform for on-site screening.

  20. Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

    Directory of Open Access Journals (Sweden)

    Igor R. Costa

    2014-12-01

    Full Text Available Essential amino acids (EAA consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS and betaine-homocysteine S-methyltransferase (BHMT diverged from the expected Tree of Life (ToL relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  1. Volatile compounds and changes in flavour-related enzymes during cold storage of high-intensity pulsed electric field- and heat-processed tomato juices.

    Science.gov (United States)

    Aguiló-Aguayo, Ingrid; Soliva-Fortuny, Robert; Martín-Belloso, Olga

    2010-08-15

    The effects of high-intensity pulsed electric field (HIPEF) processing (35 kV cm(-1) for 1500 micros, using 4 micros bipolar pulses at 100 Hz) on the production of volatile compounds and flavour-related enzymes in tomato juice were investigated and compared with those of thermal processing (90 degrees C for 30 or 60 s). Tomato juice treated by HIPEF showed lower residual lipoxygenase (LOX) activity (70.2%) than juice heated at 90 degrees C for 60 s (80.1%) or 30 s (93.2%). In contrast, hydroperoxide lyase (HPL) was almost completely inactivated when the juice was subjected to 90 degrees C for 60 s, whereas roughly 50% of the control tomato juice was depleted after HIPEF treatment or thermal processing at 90 degrees C for 30 s. A slight decrease was observed in the initial LOX activity of treated and untreated samples during storage, whereas initial HPL activity was strongly affected over time. HIPEF-treated juice exhibited higher levels of compounds contributing to tomato aroma than untreated and heat-treated juices throughout storage. Thus HIPEF processing can preserve flavour quality and stability of tomato juice compared with conventional thermal treatments. Copyright (c) 2010 Society of Chemical Industry.

  2. Potential Therapeutic Applications of Mucuna pruriens Peptide Fractions Purified by High-Performance Liquid Chromatography as Angiotensin-Converting Enzyme Inhibitors, Antioxidants, Antithrombotic and Hypocholesterolemic Agents.

    Science.gov (United States)

    Herrera-Chalé, Francisco; Ruiz-Ruiz, Jorge Carlos; Betancur-Ancona, David; Segura-Campos, Maira Rubi

    2016-02-01

    A Mucuna pruriens protein concentrate was hydrolyzed with a digestive (pepsin-pancreatin) enzymatic system. The soluble portion of the hydrolysate was fractionated by ultrafiltration and the ultrafiltered peptide fraction (PF) with lower molecular weight was purified by reversed-phase high-performance liquid chromatography. The PF obtained were evaluated by testing the biological activity in vitro. Fractions showed that the ability to inhibit the angiotensin-converting enzyme had IC50 values that ranged from 2.7 to 6.2 μg/mL. Trolox equivalent antioxidant capacity values ranged from 132.20 to 507.43 mM/mg. The inhibition of human platelet aggregation ranged from 1.59% to 11.11%, and the inhibition of cholesterol micellar solubility ranged from 0.24% to 0.47%. Hydrophobicity, size, and amino acid sequence could be factors in determining the biological activity of peptides contained in fractions. This is the first report that M. pruriens peptides act as antihypertensives, antioxidants, and inhibitors for human platelet aggregation and cholesterol micellar solubility in vitro.

  3. Pioglitazone Upregulates Angiotensin Converting Enzyme 2 Expression in Insulin-Sensitive Tissues in Rats with High-Fat Diet-Induced Nonalcoholic Steatohepatitis

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2014-01-01

    Full Text Available Background and Aim. Thiazolidinediones (TZDs can improve hepatic steatosis in nonalcoholic steatohepatitis (NASH. Angiotensin (Ang II, the primary effector of renin-angiotensin system (RAS, plays vital roles in the development and progression of NASH. And some AngII-mediated effects can be regulated by TZDs. Angiotensin-converting enzyme (ACE 2, a new component of RAS, can degrade Ang II to attenuate its subsequent physiological actions. We aimed to evaluate the effects of TZDs on ACE2 expression in insulin-sensitive tissues in NASH rats. Methods. Forty rats were divided into the normal control, high-fat diet (HFD, pioglitazone control, and HFD plus pioglitazone groups. After 24 weeks of treatment, we evaluated changes in liver histology and tissue-specific ACE2 expression. Results. ACE2 gene and protein expression was significantly greater in liver and adipose tissue in the HFD group compared with normal control group, while was significantly reduced in skeletal muscle. Pioglitazone significantly reduced the degree of hepatic steatosis compared with the HFD group. Pioglitazone significantly increased ACE2 protein expression in liver, adipose tissue, and skeletal muscle compared with the HFD group. Conclusions. Pioglitazone improves hepatic steatosis in the rats with HFD-induced NASH and upregulates ACE2 expression in insulin-sensitive tissues.

  4. A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

    Science.gov (United States)

    Li, Ye; Cassone, Vincent M

    2015-09-01

    A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Multivariate analysis and clustering reveal high morphological diversity in Tunisian autochthonous grapes (Vitis vinifera: insights into characterization, conservation and commercialization

    Directory of Open Access Journals (Sweden)

    Myriam Lamine

    2014-06-01

    Significance and impact of the study: In this investigation, we highlight the importance of importance of breeding programs, commercialization and evaluation of economically valuable characteristics of the highly diverse autochthonous grapevine cultivars from Tunisia.

  6. Discovery of novel hydroxamates as highly potent tumor necrosis factor-[alpha] converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

    Energy Technology Data Exchange (ETDEWEB)

    Mazzola Jr., Robert D.; Zhu, Zhaoning; Sinning, Lisa; McKittrick, Brian; Lavey, Brian; Spitler, James; Kozlowski, Joseph; Neng-Yang, Shih; Zhou, Guowei; Guo, Zhuyan; Orth, Peter; Madison, Vincent; Sun, Jing; Lundell, Daniel; Niu, Xiaoda (SPRI)

    2010-10-01

    A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1{prime}-S3{prime} pocket.

  7. A novel, highly conserved metallothionein family in basidiomycete fungi and characterization of two representative SlMTa and SlMTb genes in the ectomycorrhizal fungus Suillus luteus.

    Science.gov (United States)

    Nguyen, Hoai; Rineau, François; Vangronsveld, Jaco; Cuypers, Ann; Colpaert, Jan V; Ruytinx, Joske

    2017-07-01

    The basidiomycete Suillus luteus is an important member of the ectomycorrhizal community that thrives in heavy metal polluted soils covered with pioneer pine forests. This study aimed to identify potential heavy metal chelators in S. luteus. Two metallothionein (MT) coding genes, SlMTa and SlMTb, were identified. When heterologously expressed in yeast, both SlMTa and SlMTb can rescue the Cu sensitive mutant from Cu toxicity. In S. luteus, transcription of both SlMTa and SlMTb is induced by Cu but not Cd or Zn. Several putative Cu-sensing and metal-response elements are present in the promoter sequences. These results indicate that SlMTa and SlMTb function as Cu-thioneins. Homologs of the S. luteus MTs are present in 49 species belonging to 10 different orders of the subphylum Agaricomycotina and are remarkably conserved. The length of the proteins, number and distribution of cysteine residues indicate a novel family of fungal MTs. The ubiquitous and highly conserved features of these MTs suggest that they are important for basic cellular functions in species in the subphylum Agaricomycotina. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Science.gov (United States)

    Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

    2010-11-18

    The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  9. Snake venomics of Lachesis muta rhombeata and genus-wide antivenomics assessment of the paraspecific immunoreactivity of two antivenoms evidence the high compositional and immunological conservation across Lachesis.

    Science.gov (United States)

    Pla, Davinia; Sanz, Libia; Molina-Sánchez, Pedro; Zorita, Virginia; Madrigal, Marvin; Flores-Díaz, Marietta; Alape-Girón, Alberto; Núñez, Vitelbina; Andrés, Vicente; Gutiérrez, José María; Calvete, Juan J

    2013-08-26

    We report the proteomic analysis of the Atlantic bushmaster, Lachesis muta rhombeata, from Brazil. Along with previous characterization of the venom proteomes of L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. acrochorda (Colombia), and L. muta muta (Bolivia), the present study provides the first overview of the composition and distribution of venom proteins across this wide-ranging genus, and highlights the remarkable similar compositional and pharmacological profiles across Lachesis venoms. The paraspecificity of two antivenoms, produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro Picado (Costa Rica) using different conspecific taxa in the immunization mixtures, was assessed using genus-wide comparative antivenomics. This study confirms that the proteomic similarity among Lachesis sp. venoms is mirrored in their high immunological conservation across the genus. The clinical and therapeutic consequences of genus-wide venomics and antivenomics investigations of Lachesis venoms are discussed. The proteomics characterization of L. m. rhombeata venom completes the overview of Lachesis venom proteomes and confirms the remarkable toxin profile conservation across the five clades of this wide-ranging genus. Genus-wide antivenomics showed that two antivenoms, produced against L. stenophrys or L. m. rhombeata, exhibit paraspecificity towards all other congeneric venoms. Our venomics study shows that, despite the broad geographic distribution of the genus, monospecific antivenoms may achieve clinical coverage for any Lachesis sp. envenoming. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Subsets of Women With Close or Positive Margins After Breast-Conserving Surgery With High Local Recurrence Risk Despite Breast Plus Boost Radiotherapy

    International Nuclear Information System (INIS)

    Lupe, Krystine; Truong, Pauline T.; Alexander, Cheryl; Lesperance, Mary; Speers, Caroline; Tyldesley, Scott

    2011-01-01

    Purpose: (1) To examine the effect of surgical margin status on local recurrence (LR) and survival following breast-conserving therapy; (2) To identify subsets with close or positive margins with high LR risk despite whole breast radiotherapy (RT) plus boost. Methods and Materials: Subjects were 2,264 women with pT1–3, any N, M0 invasive breast cancer, treated with breast-conserving surgery and whole breast ± boost RT. Five-year Kaplan-Meier (KM) LR, breast cancer–specific and overall survival (BCSS and OS) were compared between cohorts with negative (n = 1,980), close (n = 222), and positive (n = 62) margins. LR rates were analyzed according to clinicopathologic characteristics. Multivariable Cox regression modeling and matched analysis of close/positive margin cases and negative margin controls were performed. Results: Median follow-up was 5.2 years. Boost RT was used in 92% of patients with close or positive margins. Five-year KM LR rates in the negative, close and positive margin cohorts were 1.3%, 4.0%, and 5.2%, respectively (p = 0.001). BCSS and OS were similar in the three margin subgroups. In the close/positive margin cohort, LR rates were 10.2% with age 10% despite whole breast plus boost RT. These patients should be considered for more definitive surgery.

  11. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    Science.gov (United States)

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  12. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  13. Energy conservation in nationalised transportation sector

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, R C

    1980-01-01

    About 60% of high speed diesel is consumed by the road transport industry. The hike in fuel prices calls for urgent measures to conserve diesel. The paper discusses the various measures undertaken to conserve diesel in the nationalized transport sector.

  14. Biodiversity conservation in a telecoupled world

    Directory of Open Access Journals (Sweden)

    L. Roman Carrasco

    2017-09-01

    Conservation practitioners need to adopt a global perspective on telecoupling and focus on the new conservation opportunities represented by shaping the social norms of affluent consumers in emerging and high-income economies.

  15. The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

    Directory of Open Access Journals (Sweden)

    Deng Jun-Xia

    2012-08-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71. Methods Unmodified 21 nucleotide small interfering RNAs (siRNAs and classic 2′-modified (2′-O-methylation or 2′-fluoro modification siRNAs were designed to target highly conserved 5′ untranslated region (UTR of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs. Results Transfection of rhabdomyosarcoma (RD cells with siRNAs targeting the EV71 genomic 5′ UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2′-modified siRNAs exhibited similar RNA interference (RNAi activity with dramatically increased serum stability in comparison with unmodified counterparts. Conclusion Sequences were identified within the highly conserved 5′ UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2′-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.

  16. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  17. Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

    Science.gov (United States)

    Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

    2016-02-24

    Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Evaluation of pressure tuning of enzymes

    DEFF Research Database (Denmark)

    Naghshineh, Mahsa

    and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer...... yield of enzymatic extraction limits the application of enzyme in pectin production. There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme...... (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste. It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE...

  19. SeqAPASS to evaluate conservation of high-throughput screening targets across non-mammalian species

    Science.gov (United States)

    Cell-based high-throughput screening (HTS) and computational technologies are being applied as tools for toxicity testing in the 21st century. The U.S. Environmental Protection Agency (EPA) embraced these technologies and created the ToxCast Program in 2007, which has served as a...

  20. Fermentation and addition of enzymes to a diet based on high-moisture corn, rapeseed cake, and peas improve digestibility of nonstarch polysaccharides, crude protein, and phosphorus in pigs

    DEFF Research Database (Denmark)

    Venås Jakobsen, Grethe; Jensen, Bent Borg; Knudsen, Knud Erik Bach

    2015-01-01

    on locally grown crops. Four diets were fed including a nonfermented liquid standard grower diet (Control) and 3 experimental diets based on high-moisture corn, rapeseed cake, and peas fed as nonfermented liquid feed (nFLF), fermented liquid feed (FLF), or FLF supplemented with an enzyme mixture of β...

  1. Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

    Science.gov (United States)

    Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

    2015-01-01

    Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species. © 2014 John Wiley & Sons Ltd.

  2. US/FRG joint report on the pebble bed high temperature reactor resource conservation potential and associated fuel cycle costs

    International Nuclear Information System (INIS)

    Teuchert, E.; Ruetten, H.J.; Worley, B.A.; Vondy, D.R.

    1979-11-01

    Independent analyses at ORNL and KFA have led to the general conclusion that the flexibility in design and operation of a high-temperature gas-cooled pebble-bed reactor (PBR) can result in favorable ore utilization and fuel costs in comparison with other reactor types, in particular, with light-water reactors (LWRs). Fuel reprocessign and recycle show considerable promise for reducing ore consumption, and even the PBR throwaway cycle is competitive with fuel recycle in an LWR. The best performance results from the use of highly enriched fuel. Proliferation-resistant measures can be taken using medium-enriched fuel at a modest ore penalty, while use of low-enriched fuel would incur further ore penalty. Breeding is possible but net generation of fuel at a significant rate would be expensive, becoming more feasible as ore costs increase substantially. The 233 U inventory for a breeder could be produced by prebreeders using 235 U fuel

  3. Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor

    NARCIS (Netherlands)

    W. Klootwijk (Willem); T.J. Visser (Theo); G.G.J.M. Kuiper (George)

    2002-01-01

    textabstractHuman type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all

  4. The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes.

    Science.gov (United States)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    2016-01-15

    The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for

  6. The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

    Directory of Open Access Journals (Sweden)

    Burnside Kellie L

    2009-11-01

    Full Text Available Abstract Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV, is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1 and three species of macaques (RFHVMm, RFHVMn and RFHVMf, and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively. We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus and MneRV2 (pig-tail, with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses

  7. Repeated conservation threats across the Americas: High levels of blood and bone lead in the Andean Condor widen the problem to a continental scale.

    Science.gov (United States)

    Wiemeyer, Guillermo M; Pérez, Miguel A; Torres Bianchini, Laura; Sampietro, Luciano; Bravo, Guillermo F; Jácome, N Luis; Astore, Vanesa; Lambertucci, Sergio A

    2017-01-01

    Wildlife lead exposure is an increasing conservation threat that is being widely investigated. However, for some areas of the world (e.g., South America) and certain species, research on this subject is still scarce or only local information is available. We analyzed the extent and intensity of lead exposure for a widely distributed threatened species, the Andean Condor (Vultur gryphus). We conducted the study at two different scales: 1) sampling of birds received for rehabilitation or necropsy in Argentina, and 2) bibliographic review and extensive survey considering exposure event for the species' distribution in South America. Wild condors from Argentina (n = 76) presented high lead levels consistent with both recent and previous exposure (up to 104 μg/dL blood level, mean 15.47 ± 21.21 μg/dL and up to 148.20 ppm bone level, mean 23.08 ± 31.39 ppm). In contrast, captive bred individuals -not exposed to lead contamination- had much lower lead levels (mean blood level 5.63 ± 3.08 μg/dL, and mean bone level 2.76 ± 3.06 ppm). Condors were exposed to lead throughout their entire range in continental Argentina, which represents almost sixty percent (>4000 km) of their geographical distribution. We also present evidence of lead exposure events in Chile, Ecuador, and Peru. Lead poisoning is a widespread major conservation threat for the Andean Condor, and probably other sympatric carnivores from South America. The high number and wide range of Andean Condors with lead values complement the results for the California Condor and other scavengers in North America suggesting lead poisoning is a continental threat. Urgent actions are needed to reduce this poison in the wild. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Integrating Landsat Data and High-Resolution Imagery for Applied Conservation Assessment of Forest Cover in Latin American Heterogenous Landscapes

    Science.gov (United States)

    Thomas, N.; Rueda, X.; Lambin, E.; Mendenhall, C. D.

    2012-12-01

    Large intact forested regions of the world are known to be critical to maintaining Earth's climate, ecosystem health, and human livelihoods. Remote sensing has been successfully implemented as a tool to monitor forest cover and landscape dynamics over broad regions. Much of this work has been done using coarse resolution sensors such as AVHRR and MODIS in combination with moderate resolution sensors, particularly Landsat. Finer scale analysis of heterogeneous and fragmented landscapes is commonly performed with medium resolution data and has had varying success depending on many factors including the level of fragmentation, variability of land cover types, patch size, and image availability. Fine scale tree cover in mixed agricultural areas can have a major impact on biodiversity and ecosystem sustainability but may often be inadequately captured with the global to regional (coarse resolution and moderate resolution) satellite sensors and processing techniques widely used to detect land use and land cover changes. This study investigates whether advanced remote sensing methods are able to assess and monitor percent tree canopy cover in spatially complex human-dominated agricultural landscapes that prove challenging for traditional mapping techniques. Our study areas are in high altitude, mixed agricultural coffee-growing regions in Costa Rica and the Colombian Andes. We applied Random Forests regression tree analysis to Landsat data along with additional spectral, environmental, and spatial variables to predict percent tree canopy cover at 30m resolution. Image object-based texture, shape, and neighborhood metrics were generated at the Landsat scale using eCognition and included in the variable suite. Training and validation data was generated using high resolution imagery from digital aerial photography at 1m to 2.5 m resolution. Our results are promising with Pearson's correlation coefficients between observed and predicted percent tree canopy cover of .86 (Costa

  9. Deep-ocean disposal of high-activity nuclear wastes: a conservative assessment of the seafood critical pathway

    International Nuclear Information System (INIS)

    Baxter, M.S.; Economides, B.

    1984-01-01

    This paper applies conventional 'worst-case' assumptions to modelling the effects of possible future disposal of high-activity wastes in the oceans. It otherwise uses previously published and generally accepted data to assess the possible intakes of the waste nuclides via consumption of seaweeds, molluscs, crustaceans, plankton and fish. Model-predicted intakes for critical groups generally exceed ICRP-recommended limits, with 244 Cm, 241 Am and 137 Cs being the most potentially hazardous nuclides. The various seafood consumption pathways are found to rank, in decreasing order of potential hazard, as seaweeds > molluscs > plankton > fish > crustaceans. (Auth.)

  10. Occurrence of aminoglycoside-modifying enzymes among isolates of Escherichia coli exhibiting high levels of aminoglycoside resistance isolated from Korean cattle farms.

    Science.gov (United States)

    Belaynehe, Kuastros Mekonnen; Shin, Seung Won; Hong-Tae, Park; Yoo, Han Sang

    2017-08-01

    This study investigated 247 Escherichia coli isolates collected from four cattle farms to characterize aminoglycoside-modifying enzyme (AME) genes, their plasmid replicons and transferability. Out of 247 isolates a high number of isolates (total 202; 81.78%) were found to be resistant to various antibiotics by disc diffusion. Of the 247 strains, 139 (56.3%) were resistant to streptomycin, and other antibiotic resistances followed as tetracycline (12.15%), ampicillin (7%), chloramphenicol (5.7%) and trimethoprim-sulfamethoxazole (0.8%). Among 247 isolates B1 was the predominant phylogenetic group identified comprising 151 isolates (61.1%), followed by groups A (27.9%), D (7%) and B2 (4%). Out of 139 isolates investigated for AME, 130 (93.5%) isolates carried at least one AME gene. aph3″-1a and aph3″-1b (46%) were the principal genes detected, followed by aac3-IVa (34.5%). ant2″-1a was the least detected gene (2.2%). Nine (6.5%) strains carried no AME genes. Twelve (63.2%) among 19 isolates transferred an AME gene to a recipient and aph3΄-1a was the dominant transferred gene. Transferability mainly occurred via the IncFIB replicon type (52.6%). Pulsed-field gel electrophoresis typing demonstrated a higher degree of diversity with 14 distinct cluster types. This result suggests that commensal microflora from food-producing animals has a tremendous ability to harbor and transfer AME genes, and poses a potential risk by dissemination of resistance to humans through the food chain. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

    Science.gov (United States)

    Kim, Wan-Joong; Cho, Hyo Young; Jeong, Bongjin; Byun, Sangwon; Huh, JaeDoo; Kim, Young Jun

    2017-01-01

    Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min. PMID:29278402

  12. Breast-Conservative Surgery With Close or Positive Margins: Can the Breast Be Preserved With High-Dose-Rate Brachytherapy Boost?

    International Nuclear Information System (INIS)

    Guinot, Jose Luis; Roldan, Susana; Maronas, Maria; Tortajada, Isabel; Carrascosa, Maria; Chust, Maria Luisa; Estornell, Marian; Mengual, Jose Luis; Arribas, Leoncio

    2007-01-01

    Purpose: To evaluate the likelihood of preserving the breast in women who show close or positive margins after conservative surgery for early breast carcinoma. Methods and Materials: Since 1996, 125 women with less than 5 mm or positive margins and positive separate cavity margin sampling were entered in a prospective trial with high-dose radiotherapy. A standard dose of 50 Gy to the whole breast was followed by a high-dose-rate brachytherapy application delivering 3 fractions of 4.4 Gy in 24 hours. The median follow-up was 84 months. Results: There were only seven local recurrences, with an actuarial local control rate of 95.8% at 5 years and 91.1% at 9 years. Actuarial overall and cause-specific survival rates were 92.6% and 95% at 5 years and 86.7% and 90.4% at 9 years, respectively. Late fibrosis was the most common complication, in 30% of patients, with good or excellent cosmetic results in 77%. The final result was that 95.2% of breasts were preserved. Conclusions: Close or positive-margin breast cancer can be well managed with a high-dose boost in a wide tumor bed by means of high-dose-rate brachytherapy. This technique can avoid mastectomy or poor cosmetic resection, with minimal risk of local or general failure

  13. A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

    Science.gov (United States)

    Lyda, Todd A.; Joshi, Manju B.; Andersen, John F.; Kelada, Andrew Y.; Owings, Joshua P.; Bates, Paul A.; Dwyer, Dennis M.

    2015-01-01

    Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts. PMID:25763714

  14. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

    Directory of Open Access Journals (Sweden)

    Kevin A Wilkinson

    2008-04-01

    Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

  15. The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

    Science.gov (United States)

    Ryon, J J; Fixman, E D; Houchens, C; Zong, J; Lieberman, P M; Chang, Y N; Hayward, G S; Hayward, S D

    1993-01-01

    Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid. Images PMID:8389916

  16. Genetic structure analysis of a highly inbred captive population of the African antelope Addax nasomaculatus. Conservation and management implications.

    Science.gov (United States)

    Armstrong, E; Leizagoyen, C; Martínez, A M; González, S; Delgado, J V; Postiglioni, A

    2011-01-01

    The African antelope Addax nasomaculatus is a rare mammal at high risk of extinction, with no more than 300 individuals in the wild and 1,700 captive animals distributed in zoos around the world. In this work, we combine genetic data and genealogical information to assess the structure and genetic diversity of a captive population located at Parque Lecocq Zoo (N=27), originated from only two founders. We amplified 39 microsatellites previously described in other Artiodactyls but new to this species. Seventeen markers were polymorphic, with 2-4 alleles per locus (mean=2.71). Mean expected heterozygosity (He) per locus was between 0.050 (marker ETH3) and 0.650 (marker D5S2), with a global He of 0.43. The mean inbreeding coefficient of the population computed from pedigree records of all registered individuals (N=53) was 0.222. The mean coancestry of the population was 0.298 and F(IS) index was -0.108. These results reflect the importance of an adequate breeding management on a severely bottlenecked captive population, which would benefit by the incorporation of unrelated individuals. Thanks to the successful amplification of a large number of microsatellites commonly used in domestic bovids, this study will provide useful information for the management of this population and serve as future reference for similar studies in other captive populations of this species. © 2010 Wiley-Liss, Inc.

  17. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Directory of Open Access Journals (Sweden)

    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  18. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  19. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    Science.gov (United States)

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-11-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.

  20. CT-image based conformal high-dose rate brachytherapy boost in the conservative treatment of stage I - II breast cancer - introducing the procedure

    International Nuclear Information System (INIS)

    Kubaszewska, M.; Skowronek, J.; Chichel, A.; Kanikowski, M.; Dymnicka, M.

    2008-01-01

    Aim: Breast-conserving surgery (BCS) followed by radiotherapy (RT) has become the standard treatment for the majority of patients with early breast cancer. With regard to boost technique some disagreements are found between groups that are emphasizing the value of electron boost treatment and groups pointing out the value of interstitial brachytherapy (BT) boost treatment. We present the preliminary results in treating selected patients with early-stage breast cancer using high-dose-rate brachytherapy (HD R-BT) as a boost after breast conservation therapy (BCT). Materials/Methods: Between January 2006 and August 2007, a total of 58 female patients with first and second stage breast cancer underwent BCT. This therapeutic procedure involves BCS, whole breast radiation therapy (WBRT) and additional irradiation to the tumour bed (boost) using interstitial HDR-BT via flexible implant tubes. A 10 Gy boost dose was received by all patients. The treatment planning was based on CT-guided 3D (three-dimensional) reconstruction of the surgical clips, implant tubes and critical structures localization (skin and ribs). The accuracy of tumour bed localization, the conformity of planning target volume and treated volume were analyzed. Results: The evaluations of implant parameters involved the use of: dose volume histogram (DVH), the volume encompassed by the 100% reference isodose surface (V100%), the high dose volumecalculation (V150%, V200%, V300%), the dose non-uniformity ratio (DNR), and the conformity index (COIN). Our results were as follows: the mean PTV volume, the mean high dose volume (V150%; V200%; V300%), the DNR and COIN mean value were estimated at 57.38, 42.98, 21.38, 7.90, 0.52 and 0.83 respectively. Conclusions: CT-guided 3D HDR-BT is most appropriate for planning the boost procedure after BT especially in large breast volume, in cases with a deep seated tumour bed, as well as in patients with high risk for local recurrences. This technique reduces the

  1. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2016-01-01

    Full Text Available 2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA, conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K and aspartate (D residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models.

  2. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  3. Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

    NARCIS (Netherlands)

    A.G.A. Bijvoet (Agnes); M.A. Kroos (Marian); F.R. Pieper (Frank); M. Van der Vliet (Martin); H.A. de Boer (Herman); A.T. van der Ploeg (Ans); M.Ph. Verbeet (Martin); A.J.J. Reuser (Arnold)

    1998-01-01

    textabstractGlycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective

  4. Thermomyces lanuginosus STm: a source of thermostable hydrolytic enzymes for novel application in extraction of high-quality natural rubber from Taraxacum kok-saghyz (rubber dandelion)

    Science.gov (United States)

    Hydrolytic enzymes from a newly isolated strain of the thermophilic fungus Thermomyces lanuginosus were used to extract rubber from Taraxacum kok-saghyz commonly known as rubber (or Russian or Kazak(h)) dandelion. The fungus was isolated from garden soil and identified as Thermomyces lanuginosus STm...

  5. Effects of the angiotensin-receptor blocker telmisartan on cardiovascular events in high-risk patients intolerant to angiotensin-converting enzyme inhibitors: a randomised controlled trial

    DEFF Research Database (Denmark)

    NN, NN; Yusuf, S; Teo, K

    2008-01-01

    BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors reduce major cardiovascular events, but are not tolerated by about 20% of patients. We therefore assessed whether the angiotensin-receptor blocker telmisartan would be effective in patients intolerant to ACE inhibitors with cardiovascular...

  6. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  7. Promoting household energy conservation

    International Nuclear Information System (INIS)

    Steg, Linda

    2008-01-01

    It is commonly assumed that households must change their behaviour to reduce the problems caused by increasing levels of fossil energy use. Strategies for behaviour change will be more effective if they target the most important causes of the behaviour in question. Therefore, this paper first discusses the factors influencing household energy use. Three barriers to fossil fuel energy conservation are discussed: insufficient knowledge of effective ways to reduce household energy use, the low priority and high costs of energy savings, and the lack of feasible alternatives. Next, the paper elaborates on the effectiveness and acceptability of strategies aimed to promote household energy savings. Informational strategies aimed at changing individuals' knowledge, perceptions, cognitions, motivations and norms, as well as structural strategies aimed at changing the context in which decisions are made, are discussed. This paper focuses on the psychological literature on household energy conservation, which mostly examined the effects of informational strategies. Finally, this paper lists important topics for future research

  8. The Chthonomonas calidirosea Genome Is Highly Conserved across Geographic Locations and Distinct Chemical and Microbial Environments in New Zealand's Taupō Volcanic Zone.

    Science.gov (United States)

    Lee, Kevin C; Stott, Matthew B; Dunfield, Peter F; Huttenhower, Curtis; McDonald, Ian R; Morgan, Xochitl C

    2016-06-15

    Chthonomonas calidirosea T49(T) is a low-abundance, carbohydrate-scavenging, and thermophilic soil bacterium with a seemingly disorganized genome. We hypothesized that the C. calidirosea genome would be highly responsive to local selection pressure, resulting in the divergence of its genomic content, genome organization, and carbohydrate utilization phenotype across environments. We tested this hypothesis by sequencing the genomes of four C. calidirosea isolates obtained from four separate geothermal fields in the Taupō Volcanic Zone, New Zealand. For each isolation site, we measured physicochemical attributes and defined the associated microbial community by 16S rRNA gene sequencing. Despite their ecological and geographical isolation, the genome sequences showed low divergence (maximum, 1.17%). Isolate-specific variations included single-nucleotide polymorphisms (SNPs), restriction-modification systems, and mobile elements but few major deletions and no major rearrangements. The 50-fold variation in C. calidirosea relative abundance among the four sites correlated with site environmental characteristics but not with differences in genomic content. Conversely, the carbohydrate utilization profiles of the C. calidirosea isolates corresponded to the inferred isolate phylogenies, which only partially paralleled the geographical relationships among the sample sites. Genomic sequence conservation does not entirely parallel geographic distance, suggesting that stochastic dispersal and localized extinction, which allow for rapid population homogenization with little restriction by geographical barriers, are possible mechanisms of C. calidirosea distribution. This dispersal and extinction mechanism is likely not limited to C. calidirosea but may shape the populations and genomes of many other low-abundance free-living taxa. This study compares the genomic sequence variations and metabolisms of four strains of Chthonomonas calidirosea, a rare thermophilic bacterium from

  9. Prediction of Detailed Enzyme Functions and Identification of Specificity Determining Residues by Random Forests

    Science.gov (United States)

    Nagao, Chioko; Nagano, Nozomi; Mizuguchi, Kenji

    2014-01-01

    Determining enzyme functions is essential for a thorough understanding of cellular processes. Although many prediction methods have been developed, it remains a significant challenge to predict enzyme functions at the fourth-digit level of the Enzyme Commission numbers. Functional specificity of enzymes often changes drastically by mutations of a small number of residues and therefore, information about these critical residues can potentially help discriminate detailed functions. However, because these residues must be identified by mutagenesis experiments, the available information is limited, and the lack of experimentally verified specificity determining residues (SDRs) has hindered the development of detailed function prediction methods and computational identification of SDRs. Here we present a novel method for predicting enzyme functions by random forests, EFPrf, along with a set of putative SDRs, the random forests derived SDRs (rf-SDRs). EFPrf consists of a set of binary predictors for enzymes in each CATH superfamily and the rf-SDRs are the residue positions corresponding to the most highly contributing attributes obtained from each predictor. EFPrf showed a precision of 0.98 and a recall of 0.89 in a cross-validated benchmark assessment. The rf-SDRs included many residues, whose importance for specificity had been validated experimentally. The analysis of the rf-SDRs revealed both a general tendency that functionally diverged superfamilies tend to include more active site residues in their rf-SDRs than in less diverged superfamilies, and superfamily-specific conservation patterns of each functional residue. EFPrf and the rf-SDRs will be an effective tool for annotating enzyme functions and for understanding how enzyme functions have diverged within each superfamily. PMID:24416252

  10. Geographies of Conservation I: De-extinction and Precision Conservation

    OpenAIRE

    Adams, William Mark

    2016-01-01

    Extinction has long been a central concern in biodiversity conservation. Today, de-extinction offers interesting possibilities of restoring charismatic species and ecosystem function, but also risks and costs. Most de-extinction depends on genetic engineering and synthetic biology. These technologies are also proposed for use in ‘gene tweaking’ in wild species to enhance their chance of survival. Within conservation, the resulting debates pit an optimistic world of high-tech ‘precision con...

  11. Cytosolic malic enzyme 1 (ME1 mediates high fat diet-induced adiposity, endocrine profile, and gastrointestinal tract proliferation-associated biomarkers in male mice.

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Dwairi

    Full Text Available Obesity and associated hormonal disturbances are risk factors for colon cancer. Cytosolic Malic Enzyme (ME1 generates NADPH used for lipogenesis in gastrointestinal (GI, liver and adipose tissues. We have reported that inclusion of soy protein isolate (SPI in the diet lowered body fat content and colon tumor incidence of rats fed AIN-93G diet, while others have demonstrated SPI inhibition of rat hepatic ME1 expression. The present study examined the individual and combined effects of dietary SPI and absence of ME1 on: 1 serum concentrations of hormones implicated in colon cancer development, 2 expression of lipogenic and proliferation-associated genes in the mouse colon and small intestine, and 3 liver and adipose expression of lipogenic and adipocytokine genes that may contribute to colon cancer predisposition.Weanling wild type (WT and ME1 null (MOD-1 male mice were fed high-fat (HF, iso-caloric diets containing either casein (CAS or SPI as sole protein source for 5 wks. Somatic growth, serum hormone and glucose levels, liver and adipose tissue weights, GI tissue parameters, and gene expression were evaluated.The MOD-1 genotype and SPI-HF diet resulted in decreases in: body and retroperitoneal fat weights, serum insulin, serum leptin, leptin/adiponectin ratio, adipocyte size, colon mTOR and cyclin D1 mRNA abundance, and jejunum FASN mRNA abundance, when compared to WT mice fed CAS-HF. Regardless of diet, MOD-1 mice had reductions in liver weight, liver steatosis, and colon crypt depth, and increases in adipose tissue expression of IRS1 and IRS2, compared to WT mice. SPI-HF diet reduced ME1 gene expression only in retroperitoneal fat.Data suggest that the pharmacological targeting of ME1 or the inclusion of soy protein in the diet may provide avenues to reduce obesity and its associated pro-tumorigenic endocrine environment and improve insulin sensitivity, potentially disrupting the obesity-colon cancer connection.

  12. Evaluation the consistency of location of moist desquamation and skin high dose area for breast cancer patients receiving adjuvant radiotherapy after breast conservative surgery

    International Nuclear Information System (INIS)

    Sun, Li-Min; Huang, Eng-Yen; Liang, Ji-An; Meng, Fan-Yun; Chang, Gia-Hsin; Tsao, Min-Jen

    2013-01-01

    To evaluate whether the location of moist desquamation matches high dose area for breast cancer patients receiving adjuvant radiotherapy (RT) after breast conservative surgery. One hundred and nine breast cancer patients were enrolled to this study. Their highest skin dose area (the hot spot) was estimated from the treatment planning. We divided the irradiated field into breast; sternal/parasternal; axillary; and inframammary fold areas. The location for moist desquamation was recorded to see if it matches the hot spot. We also analyzed other possible risk factors which may be related to the moist desquamation. Forty-eight patients with 65 locations developed moist desquamation during the RT course. Patients with larger breast sizes and easy to sweat are two independent risk factors for moist desquamation. The distribution of moist desquamation occurred most in the axillary area. All nine patients with the hot spots located at the axillary area developed moist desquamation at the axillary area, and six out of seven patients with the hot spots located at the inframammary fold developed moist desquamation there. The majority of patients with moist desquamation over the breast or sternal/parasternal areas had the hot spots located at these areas. For a patient with moist desquamation, if a hot spot is located at the axillary or inframammary fold areas, it is very likely to have moist desquamation occur there. On the other hand, if moist desquamation occurs over the breast or sternal/parasternal areas, we can highly expect these two areas are also the hot spot locations

  13. ENZYME RESISTANCE OF GENETICALLY MODIFIED STARCH POTATOES

    Directory of Open Access Journals (Sweden)

    A. Sh. Mannapova

    2015-01-01

    Full Text Available Here in this article the justification of expediency of enzyme resistant starch use in therapeutic food products is presented . Enzyme resistant starch is capable to resist to enzymatic hydrolysis in a small intestine of a person, has a low glycemic index, leads to decrease of postprandial concentration of glucose, cholesterol, triglycerides in blood and insulin reaction, to improvement of sensitivity of all organism to insulin, to increase in sense of fulness and to reduction of adjournment of fats. Resistant starch makes bifidogenшс impact on microflora of a intestine of the person, leads to increase of a quantity of lactobacillus and bifidobacterium and to increased production of butyric acid in a large intestine. In this regard the enzyme resistant starch is an important component in food for prevention and curing of human diseases such as diabetes, obesity, colitis, a cancer of large and direct intestine. One method is specified by authors for imitation of starch digestion in a human body. This method is based on the definition of an enzyme resistance of starch in vitro by its hydrolysis to glucose with application of a glucoamylase and digestive enzyme preparation Pancreatin. This method is used in researches of an enzyme resistance of starch, of genetically modified potato, high amylose corn starch Hi-Maize 1043 and HYLON VII (National Starch Food Innovation, USA, amylopectin and amylose. It is shown that the enzyme resistance of the starch emitted from genetically modified potatoes conforms to the enzyme resistance of the high amylose corn starch “Hi-Maize 1043 and HYLON VII starch”, (National Starch Food Innovation, the USA relating to the II type of enzyme resistant starch. It is established that amylopectin doesn't have the enzyme resistant properties. The results of researches are presented. They allow us to make the following conclusion: amylose in comparison with amylopectin possesses higher enzyme resistance and gives to

  14. Lignocellulose biotechnology: issues of bioconversion and enzyme ...

    African Journals Online (AJOL)

    Lignocellulose biotechnology: issues of bioconversion and enzyme production. ... and secondly to highlight some of the modern approaches which potentially could be used to tackle one of the major impediments, namely high enzyme cost, to speed-up the extensive commercialisation of the lignocellulose bioprocessing.

  15. It's not too late for the harpy eagle (Harpia harpyja: high levels of genetic diversity and differentiation can fuel conservation programs.

    Directory of Open Access Journals (Sweden)

    Heather R L Lerner

    2009-10-01

    Full Text Available The harpy eagle (Harpia harpyja is the largest Neotropical bird of prey and is threatened by human persecution and habitat loss and fragmentation. Current conservation strategies include local education, captive rearing and reintroduction, and protection or creation of trans-national habitat blocks and corridors. Baseline genetic data prior to reintroduction of captive-bred stock is essential for guiding such efforts but has not been gathered previously.We assessed levels of genetic diversity, population structure and demographic history for harpy eagles using samples collected throughout a large portion of their geographic distribution in Central America (n = 32 and South America (n = 31. Based on 417 bp of mitochondrial control region sequence data, relatively high levels of haplotype and nucleotide diversity were estimated for both Central and South America, although haplotype diversity was significantly higher for South America. Historical restriction of gene flow across the Andes (i.e. between our Central and South American subgroups is supported by coalescent analyses, the haplotype network and significant F(ST values, however reciprocally monophyletic lineages do not correspond to geographical locations in maximum likelihood analyses. A sudden population expansion for South America is indicated by a mismatch distribution analysis, and further supported by significant (p<0.05 negative values of Fu and Li's D(F and F, and Fu's F(S. This expansion, estimated at approximately 60 000 years BP (99 000-36 000 years BP 95% CI, encompasses a transition from a warm and dry time period prior to 50 000 years BP to an interval of maximum precipitation (50 000-36 000 years BP. Notably, this time period precedes the climatic and habitat changes associated with the last glacial maximum. In contrast, a multimodal distribution of haplotypes was observed for Central America suggesting either population equilibrium or a recent decline.High levels of

  16. Non-native and native organisms moving into high elevation and high latitude ecosystems in an era of climate change: new challenges for ecology and conservation

    Science.gov (United States)

    Pauchard, Aníbal; Albihn, Ann; Alexander, Jake; Burgess, Treena; Daehler, Curt; Essl, Franz; Evengard, Birgitta; Greenwood, Greg; Haider, Sylvia; Lenoir, Jonathan; McDougall, K.; Milbau, Ann; Muths, Erin L.; Nunez, Martin; Pellissier, Lois; Rabitsch, Wolfgang; Rew, Lisa; Robertson, Mark; Sanders, Nathan; Kueffer, Christoph

    2016-01-01

    Cold environments at high elevation and high latitude are often viewed as resistant to biological invasions. However, climate warming, land use change and associated increased connectivity all increase the risk of biological invasions in these environments. Here we present a summary of the key discussions of the workshop ‘Biosecurity in Mountains and Northern Ecosystems: Current Status and Future Challenges’ (Flen, Sweden, 1–3 June 2015). The aims of the workshop were to (1) increase awareness about the growing importance of species expansion—both non-native and native—at high elevation and high latitude with climate change, (2) review existing knowledge about invasion risks in these areas, and (3) encourage more research on how species will move and interact in cold environments, the consequences for biodiversity, and animal and human health and wellbeing. The diversity of potential and actual invaders reported at the workshop and the likely interactions between them create major challenges for managers of cold environments. However, since these cold environments have experienced fewer invasions when compared with many warmer, more populated environments, prevention has a real chance of success, especially if it is coupled with prioritisation schemes for targeting invaders likely to have greatest impact. Communication and co-operation between cold environment regions will facilitate rapid response, and maximise the use of limited research and management resources.

  17. Targeted enzyme prodrug therapies.

    Science.gov (United States)

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  18. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  19. Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide.

    Science.gov (United States)

    Kretova, Olga V; Fedoseeva, Daria M; Gorbacheva, Maria A; Gashnikova, Natalya M; Gashnikova, Maria P; Melnikova, Nataliya V; Chechetkin, Vladimir R; Kravatsky, Yuri V; Tchurikov, Nickolai A

    2017-09-15

    RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%-97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma

    DEFF Research Database (Denmark)

    Arshid, Samina; Tahir, Muhammad; Fontes, Belchor

    2017-01-01

    and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using...... functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome...... degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal...

  1. HYPOLIPIDEMIC EFFECT OF CURCUMIN OR CO-ENZYME Q1-0 AND THEIR MIXTURE ON OBESE RATS FED A HIGH CHOLESTEROL DIET

    International Nuclear Information System (INIS)

    SHAHIN, M.I.M.

    2008-01-01

    In the current study, hyperlipidemia was induced in the rats by feeding diet enriched with cholesterol for two weeks. After 2 weeks of induction of hypercholesterolemia in rats and in comparison to normal rats, the results showed that incorporation of extra cholesterol in diet led to significant increases in serum cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, leptin and MDA levels. On the other hand, total serum triiodothyronine (T3), liver glutathione (GSH) and glutathione peroxidase (GPx) activities were decreased significantly in cholesterol fed rats. The concentration of TBARS in the liver was elevated.All previous parameters were corrected after the hypercholesterolemic rats were treated with curcumin or co-enzyme Q 1 -0 or a mixture of them dependent on the time of treatment. These findings are consistent with the concept that curcumin and co-enzyme Q 10 are antioxidant agents. The underlying mechanisms of these effects were discussed

  2. Making conservation work for everyone

    Energy Technology Data Exchange (ETDEWEB)

    Wiersma, J. [Veridian Corp., Ajax, ON (Canada)

    2004-07-01

    This presentation discussed the economic value of conservation, the optimal deployment of energy conservation. A sample load profile was presented to demonstrate how much electricity the average residential customer uses on a summer day. The average customer does not have the tools to understand the financial consequences of conservation for different types of equipment at different times of the day. Smart metering technology could help in this regard. Accurate unsubsidized prices are also considered to be the best incentive to conserve because customers will reduce electricity use when the prices are high. It was also suggested that standards for new appliances should be increased effectively to their economic value. The enablers to energy conservation include solid consumer education programs, real time metering in places where it is cost effective, real time pricing in places where it is practical, and power rates that reflect real costs. Barriers to energy conservation include the residual economic advantage that may be insufficient to justify investment; support from local distribution companies and transmission companies if the lost revenue adjustment mechanism (LRAM) is not sufficient to recover lost revenue and if LDCs are not sufficiently involved in the design of the electricity conservation program. 7 figs.

  3. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    Science.gov (United States)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  4. Enzymes of industrial purpose - review of the market of enzyme preparations and prospects for its development

    Directory of Open Access Journals (Sweden)

    A. A. Tolkacheva

    2017-01-01

    Full Text Available Microbial enzyme preparations are increasingly replacing conventional chemical catalysts in a number of industrial processes. Such drugs, in addition to environmental friendliness and high activity, have a number of advantages over enzyme preparations of vegetable and animal origin, namely: the production of microbial enzymes in bioreactors is easily controlled and predictable; excreted microbiological enzymes are more stable than intracellular animals and plant enzymes; the genetic diversity of microorganisms makes it possible to produce enzyme preparations with a wide range of specificity; microbiological enzymes can be synthesized year-round, in contrast to the production of plant enzymes, which is often seasonal. The leaders of the world market of enzymes are proteases and amylases, which account for 25% and 15%, respectively. Over the past five years, the world market for carbohydrases, including mainly amylases, cellulases and xylanases, has been the fastest growing segment of the enzyme market with an aggregate annual growth rate of more than 7.0%. Another major product of the industrial enzyme market, which has a great potential for growth, is lipases. From the point of view of designation, the main part is represented by food and food enzymes. The Russian market continues to be unsaturated - the current supply is not able to meet the needs of the Russian feed and food industry in enzyme preparations. Enzyme preparations of domestic producers are in demand in forage production, while food industrial enterprises prefer imported products. The most significant enterprises in the enzymatic industry in Russia at the moment are Sibbiofarm, AgroSistema, Agroferment. In the light of the Russian policy of increasing food security, the development of the domestic enzyme industry is an extremely topical task.

  5. IAA-Ala Resistant3, an evolutionarily conserved target of miR167, mediates Arabidopsis root architecture changes during high osmotic stress

    KAUST Repository

    Kinoshita, Natsuko

    2012-09-01

    The functions of microRNAs and their target mRNAs in Arabidopsis thaliana development have been widely documented; however, roles of stress-responsive microRNAs and their targets are not as well understood. Using small RNA deep sequencing and ATH1 microarrays to profile mRNAs, we identified IAA-Ala Resistant3 (IAR3) as a new target of miR167a. As expected, IAR3 mRNA was cleaved at the miR167a complementary site and under high osmotic stress miR167a levels decreased, whereas IAR3 mRNA levels increased. IAR3 hydrolyzes an inactive form of auxin (indole-3-acetic acid [IAA]-alanine) and releases bioactive auxin (IAA), a central phytohormone for root development. In contrast with the wild type, iar3 mutants accumulated reduced IAA levels and did not display high osmotic stress-induced root architecture changes. Transgenic plants expressing a cleavage-resistant form of IAR3 mRNA accumulated high levels of IAR3 mRNAs and showed increased lateral root development compared with transgenic plants expressing wild-type IAR3. Expression of an inducible noncoding RNA to sequester miR167a by target mimicry led to an increase in IAR3 mRNA levels, further confirming the inverse relationship between the two partners. Sequence comparison revealed the miR167 target site on IAR3 mRNA is conserved in evolutionarily distant plant species. Finally, we showed that IAR3 is required for drought tolerance. © 2012 American Society of Plant Biologists. All rights reserved.

  6. IAA-Ala Resistant3, an evolutionarily conserved target of miR167, mediates Arabidopsis root architecture changes during high osmotic stress

    KAUST Repository

    Kinoshita, Natsuko; Wang, Huan; Kasahara, Hiroyuki; Liu, Jun; MacPherson, Cameron R.; Machida, Yasunori; Kamiya, Yuji; Hannah, Matthew A.; Chuaa, Nam Hai

    2012-01-01

    The functions of microRNAs and their target mRNAs in Arabidopsis thaliana development have been widely documented; however, roles of stress-responsive microRNAs and their targets are not as well understood. Using small RNA deep sequencing and ATH1 microarrays to profile mRNAs, we identified IAA-Ala Resistant3 (IAR3) as a new target of miR167a. As expected, IAR3 mRNA was cleaved at the miR167a complementary site and under high osmotic stress miR167a levels decreased, whereas IAR3 mRNA levels increased. IAR3 hydrolyzes an inactive form of auxin (indole-3-acetic acid [IAA]-alanine) and releases bioactive auxin (IAA), a central phytohormone for root development. In contrast with the wild type, iar3 mutants accumulated reduced IAA levels and did not display high osmotic stress-induced root architecture changes. Transgenic plants expressing a cleavage-resistant form of IAR3 mRNA accumulated high levels of IAR3 mRNAs and showed increased lateral root development compared with transgenic plants expressing wild-type IAR3. Expression of an inducible noncoding RNA to sequester miR167a by target mimicry led to an increase in IAR3 mRNA levels, further confirming the inverse relationship between the two partners. Sequence comparison revealed the miR167 target site on IAR3 mRNA is conserved in evolutionarily distant plant species. Finally, we showed that IAR3 is required for drought tolerance. © 2012 American Society of Plant Biologists. All rights reserved.

  7. Mistaken identity: activating conservative political identities induces "conservative" financial decisions.

    Science.gov (United States)

    Morris, Michael W; Carranza, Erica; Fox, Craig R

    2008-11-01

    Four studies investigated whether activating a social identity can lead group members to choose options that are labeled in words associated with that identity. When political identities were made salient, Republicans (but not Democrats) became more likely to choose the gamble or investment option labeled "conservative." This shift did not occur in a condition in which the same options were unlabeled. Thus, the mechanism underlying the effect appears to be not activated identity-related values prioritizing low risk, but rather activated identity-related language (the group label "conservative"). Indeed, when political identities were salient, Republicans favored options labeled "conservative" regardless of whether the options were low or high risk. Finally, requiring participants to explain the label "conservative" before making their choice did not diminish the effect, which suggests that it does not merely reflect inattention to content or construct accessibility. We discuss the implications of these results for the literatures on identity, priming, choice, politics, and marketing.

  8. Sequence of cDNAs for mammalian H2A. Z, an evolutionarily diverged but highly conserved basal histone H2A isoprotein species

    Energy Technology Data Exchange (ETDEWEB)

    Hatch, C L; Bonner, W M

    1988-02-11

    The nucleotide sequences of cDNAs for the evolutionarily diverged but highly conserved basal H2A isoprotein, H2A.Z, have been determined for the rat, cow, and human. As a basal histone, H2A.Z is synthesized throughout the cell cycle at a constant rate, unlinked to DNA replication, and at a much lower rate in quiescent cells. Each of the cDNA isolates encodes the entire H2A.Z polypeptide. The human isolate is about 1.0 kilobases long. It contains a coding region of 387 nucleotides flanked by 106 nucleotides of 5'UTR and 376 nucleotides of 3'UTR, which contains a polyadenylation signal followed by a poly A tail. The bovine and rat cDNAs have 97 and 94% nucleotide positional identity to the human cDNA in the coding region and 98% in the proximal 376 nucleotides of the 3'UTR which includes the polyadenylation signal. A potential stem-forming sequence imbedded in a direct repeat is found centered at 261 nucleotides into the 3'UTR. Each of the cDNA clones could be transcribed and translated in vitro to yield H2A.Z protein. The mammalian H2A.Z cDNA coding sequences are approximately 80% similar to those in chicken and 75% to those in sea urchin.

  9. Particle-number conserving analysis for the 2-quasiparticle and high-K multi-quasiparticle states in doubly-odd 174,176Lu

    International Nuclear Information System (INIS)

    Li Bingheng; Lei Yi'an; Zhang Zhenhua

    2013-01-01

    Two-quasiparticle bands and low-lying excited high-K four-, six-, and eight-quasiparticle bands in the doubly-odd 174,176 Lu are analyzed by using the cranked shell model (CSM) with the pairing correlations treated by a particle-number conserving (PNC) method, in which the blocking effects are taken into account exactly. The proton and neutron Nilsson level schemes for 174,176 Lu are taken from the adjacent odd-A Lu and Hf isotopes, which are adopted to reproduce the experimental bandhead energies of the one-quasiproton and one-quasineutron bands of these odd-A Lu and Hf nuclei, respectively. Once the quasiparticle configurations are determined, the experimental bandhead energies and the moments of inertia of these two- and multi-quasiparticle bands are well reproduced by PNC-CSM calculations. The Coriolis mixing of the low-K (K=|Ω 1 -Ω 2 |) two-quasiparticle band of the Gallagher-Moszkowski doublet with one nucleon in the Ω=1/2 orbital is analyzed. (authors)

  10. Conservation potential of agricultural water conservation subsidies

    Science.gov (United States)

    Huffaker, Ray

    2008-07-01

    A current policy subsidizes farmers to invest in improved on-farm irrigation efficiency, expecting water to be conserved off farm. Contrary to expectation, water has been increasingly depleted in some regions after such improvements. This paper investigates the policy's failure to conserve water consistently by (1) formulating an economic model of irrigated crop production to determine a profit-maximizing irrigator's range of responses to a subsidy and (2) embedding these responses into hypothetical streamflow diagrams to ascertain their potential to conserve water under various hydrologic regimes. Testable hypotheses are developed to predict the conservation potential of a subsidy in real-world application.

  11. Method of localization and implantation of the lumpectomy site for high dose rate brachytherapy after conservative surgery for T1 and T2 breast cancer

    International Nuclear Information System (INIS)

    Perera, F.; Chisela, F.; Engel, J.; Venkatesan, V.

    1995-01-01

    Purpose: This article describes our technique of localization and implantation of the lumpectomy site of patients with T1 and T2 breast cancer. Our method was developed as part of our Phase I/II pilot study of high dose rate (HDR) brachytherapy alone after conservative surgery for early breast cancer. Methods and Materials: In March 1992, we started a pilot study of HDR brachytherapy to the lumpectomy site as the sole radiotherapy after conservative surgery for clinical T1 or T2 invasive breast cancer. Initially, the protocol required intraoperative placement of the interstitial needles at the time of definitive surgery to the breast. The protocol was then generalized to allow the implantation of the lumpectomy site after definitive surgery to the breast, either at the time of subsequent axillary nodal dissection or postoperatively. To date, five patients have been implanted intraoperatively at the time of definitive breast surgery. Twelve patients were implanted after definitive breast surgery, with 7 patients being done at the time of axillary nodal dissection and 5 patients postoperatively. We devised a method of accurately localizing and implanting the lumpectomy site after definitive breast surgery. The method relies on the previous placement of surgical clips by the referring surgeon to mark the lumpectomy site. For each patient, a breast mold is made with radio-opaque angiocatheters taped onto the mold in the supero-inferior direction. A planning CT scan is then obtained through the lumpectomy site. The volume of the lumpectomy site, the number of implant planes necessary, and the orientation of the implants are then determined from the CT scan. The angiocatheters provide a reference grid on the CT films to locate the entry and exit points of the interstitial needles on the plastic mold. The entry and exit points for reference needles are then transferred onto the patient's skin enabling implantation of the lumpectomy site. Needle positions with respect to

  12. Production of cellulolytic enzymes from ascomycetes

    DEFF Research Database (Denmark)

    Hansen, Gustav Hammerich; Lübeck, Mette; Frisvad, Jens Christian

    2015-01-01

    Optimizing production of cellulose degrading enzymes is of great interest in order to increase the feasibility of constructing biorefinery facilities for a sustainable supply of energy and chemical products. The ascomycete phylum has a large potential for the production of cellulolytic enzymes....... Although numerous enzymatic profiles have already been unraveled, the research has been covering only a limited number of species and genera, thus leaving many ascomycetes to be analyzed. Such analysis requires choosing appropriate media and cultivation methods that ensure enzyme profiles with high...... specificities and activities. However, the choice of media, cultivation methods and enzyme assays highly affect the enzyme activity profile observed. This review provides an overview of enzymatic profiles for several ascomycetes covering phylogenetically distinct genera and species. The profiles of cellulose...

  13. Transcriptional regulation of the xylanolytic enzyme system of Aspergillus

    NARCIS (Netherlands)

    Peij, van N.N.M.E.

    1999-01-01

    Filamentous fungi, such as Aspergillus niger , produce high levels of polysaccharide degrading enzymes and are frequently used as production organisms for industrial enzyme preparations. The application of these polysaccharidases as xylanases and cellulases comprises

  14. [Phylogenetic diversity and cold-adaptive hydrolytic enzymes of culturable psychrophilic bacteria associated with sea ice from high latitude ocean, Artic].

    Science.gov (United States)

    Yu, Yong; Li, Hui-Rong; Chen, Bo; Zeng, Yin-Xin; He, Jian-Feng

    2006-04-01

    The phylogenetic diversity of culturable psychrophilic bacteria associated with sea ice from high latitude sea (77 degrees 30'N - 81 degrees 12'N), Canadian Basin and Greenland sea Arctic, was investigated. A total of 37 psychrophilic strains were isolated using three different methods of ( i ) spread plate method: 100 microL of each dilution ice-melt sample was spreaded onto the surface of Marine 2216 agar (DIFCO laboratories, Detroit, MI) and incubated for 2 to 6 weeks at 4 degrees C; ( ii ) bath culture and spread plate method: 1 mL of sample was added to 9mL of NSW (unamended natural seawater, 0.2 microm prefiltered and autoclaved) and incubated for 1 months at - 1 degrees C, then spread plate method was used to isolate bacterial strains from the pre-cultured samples; ( iii ) cold shock, bath culture and spread plate method: samples were exposed to - 20 degrees C for 24h, then bacterial strains isolated by bath culture and spread plate method under aerobic conditions. Nearly half of psychrophilic strains are isolated by using method iii . 16S rDNA nearly full-length sequence analysis reveal that psychrophilic strains fall in two phylogenetic divisions, gamma-proteobacteria (in the genera Colwellia, Marinobacter, Shewanella, Thalassomonas, Glaciecola, Marinomonas and Pseudoalteromonas) and Cytophaga-Flexibacter-Bacteroides (in the genera Flavobacterium and Psychroflexus). Nine of bacterial isolates (BSi20007, BSi20497, BSi20517, BSi20537, BSi20170, BSi20001, BSi20002, BSi20675 and BSi20101) quite likely represent novel species (16S rDNA sequence similarity below 97%). One of strains (BSi20002) from Canadian Basin shows 100% sequence similarity to the Antarctic Weddell sea ice isolate Marinobacter sp. ANT8277, suggesting bacteria may have a bipolar distribution at the species level. AF283859 sequences were submitted to the BLAST search program of the National Center for Biotechnology Information website (NCBI, http://www. ncbi. nlm.nih. gov). Twenty sequences

  15. Conservation and genetic characterisation of common bean landraces from Cilento region (southern Italy): high differentiation in spite of low genetic diversity.

    Science.gov (United States)

    De Luca, Daniele; Cennamo, Paola; Del Guacchio, Emanuele; Di Novella, Riccardo; Caputo, Paolo

    2018-02-01

    Since its introduction from Central-South America to Italy almost 500 years ago, the common bean (Phaseolus vulgaris L.) was largely cultivated across the peninsula in hundreds of different landraces. However, globalisation and technological modernisation of agricultural practices in the last decades promoted the cultivation of few varieties at the expense of traditional and local agro-ecotypes, which have been confined to local markets or have completely disappeared. The aim of this study was to evaluate the genetic diversity and differentiation in 12 common bean landraces once largely cultivated in the Cilento region (Campania region, southern Italy), and now the object of a recovery program to save them from extinction. The analysis conducted using 13 nuclear microsatellite loci in 140 individuals revealed a high degree of homozygosity within each landrace and a strong genetic differentiation that was reflected in the success in assigning individuals to the source landrace. On the contrary, internal transcribed spacers 1 and 2, analysed in one individual per landrace, were highly similar among common bean landraces but allowed the identification of a cowpea variety (Vigna unguiculata Walp.), a crop largely cultivated in the Old World before the arrival of common bean from Americas. In conclusion, our study highlighted that conservation of landraces is important not only for the cultural and socio-economic value that they have for local communities, but also because the time and conditions in which they have been selected have led to that genetic distinctiveness that is at the basis of many potential agronomical applications and dietary benefits.

  16. The albinism of the feral Asinara white donkeys (Equus asinus) is determined by a missense mutation in a highly conserved position of the tyrosinase (TYR) gene deduced protein.

    Science.gov (United States)

    Utzeri, V J; Bertolini, F; Ribani, A; Schiavo, G; Dall'Olio, S; Fontanesi, L

    2016-02-01

    A feral donkey population (Equus asinus), living in the Asinara National Park (an island north-west of Sardinia, Italy), includes a unique white albino donkey subpopulation or colour morph that is a major attraction of this park. Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In this study, we analysed the donkey TYR gene as a strong candidate to identify the causative mutation of the albinism of these donkeys. The TYR gene was sequenced from 13 donkeys (seven Asinara white albino and six coloured animals). Seven single nucleotide polymorphisms were identified. A missense mutation (c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across kingdoms), which disrupts the first copper-binding site (CuA) of functional protein, was identified in the homozygous condition (G/G or D/D) in all Asinara white albino donkeys and in the albino son of a trio (the grey parents had genotype C/G or H/D), supporting the recessive mode of inheritance of this mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had genotype G/G whereas all other coloured donkeys had genotype C/C or C/G. Across-population association between the c.604C>G genotypes and the albino coat colour was highly significant (P = 6.17E-18). The identification of the causative mutation of the albinism in the Asinara white donkeys might open new perspectives to study the dynamics of this putative deleterious allele in a feral population and to manage this interesting animal genetic resource. © 2015 Stichting International Foundation for Animal Genetics.

  17. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  18. Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms.

    Directory of Open Access Journals (Sweden)

    Deanne W Sammond

    Full Text Available Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.

  19. Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms.

    Science.gov (United States)

    Sammond, Deanne W; Kastelowitz, Noah; Himmel, Michael E; Yin, Hang; Crowley, Michael F; Bomble, Yannick J

    2016-01-01

    Understanding how proteins adapt to function at high