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Sample records for broad substrate specificity

  1. Microbial transglutaminase displays broad acyl-acceptor substrate specificity

    DEFF Research Database (Denmark)

    T. Gundersen, Maria; Keillor, Jeffrey W.; Pelletier, Joelle N.

    2013-01-01

    , and Trp—but not Ile—also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group—particularly if aromatic—was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly...... understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG.We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys...

  2. Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

    OpenAIRE

    Chu, Yindi; Zhang, Zhenfeng; Wang, Qian; Luo, Yuanming; Huang, Li

    2012-01-01

    Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate prot...

  3. Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket.

    Science.gov (United States)

    Gajda, Anna D; Pawełczak, Małgorzata; Drag, Marcin

    2012-05-01

    Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized.

  4. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

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    Stefano Pascarella

    2012-01-01

    Full Text Available Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  5. Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

    Science.gov (United States)

    Sirobhushanam, Sirisha; Galva, Charitha; Sen, Suranjana; Wilkinson, Brian J; Gatto, Craig

    2016-09-01

    Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis. PMID:27320015

  6. LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Deli, Alexandra; Koutsioulis, Dimitrios; Fadouloglou, Vasiliki E.; Spiliotopoulou, Panagiota; Balomenou, Stavroula; Arnaouteli, Sofia; Tzanodaskalaki, Maria; Mavromatis, Konstantinos; Kokkinidis, Michalis; Bouriotis, Vassilis

    2010-05-19

    The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc2) and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-D-glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc2, GlcNAc3 and GlcNAc4), as well as GlcNAc-1P, N-acetyl-d-glucosamine-1 phosphate; GlcNAc-6P, N-acetyl-d-glucosamine-6 phosphate; GalNAc, N-acetyl-d-galactosamine; ManNAc, N-acetyl-d-mannosamine; UDP-GlcNAc, uridine 5'-diphosphate N-acetyl-d-glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N-acetyl-d-glucosaminyl-(β-1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine (GMDP) or N-acetyl-d-GlcN-Nα1-6-d-myo-inositol-1-HPO4-octadecyl (GlcNAc-I-P-C18). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting

  7. A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids.

    Science.gov (United States)

    Lindenkamp, Nicole; Schürmann, Marc; Steinbüchel, Alexander

    2013-09-01

    In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16∆pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating

  8. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

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    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  9. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

    Science.gov (United States)

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  10. Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation: DETAILED STRUCTURAL AND BIOCHEMICAL ANALYSIS USING TETRAPEPTIDE LIBRARY.

    Science.gov (United States)

    Reddi, Ravikumar; Saddanapu, Venkateshwarlu; Chinthapalli, Dinesh Kumar; Sankoju, Priyanka; Sripadi, Prabhakar; Addlagatta, Anthony

    2016-09-23

    Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel. PMID:27484799

  11. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Johansson, K.; Ramaswamy, S.; Ljungcrantz, C.;

    2001-01-01

    kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates......; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases....

  12. Broad Band Antireflection Coatings for Silicon and Germanium Substrates.

    Science.gov (United States)

    Bezuidenhout, Dirk Francois

    Infrared antireflection coatings for silicon and germanium substrates and some of the associated problems are addressed in this thesis. One of the first problems identified and investigated was that of the adhesion of ZnS films to germanium substrates. The cleaning of the Ge discs was evaluated by means of Auger spectroscopy. The main contaminant species found were carbon, oxygen and in the case of germanium substrates sulphur. No sulphur was found on silicon substrates. A wash in a series of organic solutions followed by a bake inside the vacuum chamber lead to much improved though still not acceptable adhesion of ZnS films to germanium substrates. The influence of a contact layer between the substrate and ZnS was investigated. Firstly, metal contact layers (Ni, Cr, Cu) were tried to improve the adhesion of the ZnS films. These samples (germanium-metal-zinc sulphide) were annealed in air in order to transfer the germanium -metal film to a germanide region and thus high optical transmission at long wave-lengths. Slight absorption still results even after the annealing of these samples. A dielectric material, Y_2O_3 , was therefore tested replacing the metal films. The system Ge-Y_2O_3 -ZnS in conjunction with an organic wash and vacuum bake lead to excellent adhesion of the ZnS layers to the germanium substrates. The next problem area addressed was that of a low refractive index material replacement for ThF _4. Four materials were investigated, i.e. ZnS, PbF_2, Y_2O _3 and YF_3. The refractive indices found for these compounds in thin film form at a wavelength of 10 μm is 2,18 for ZnS, 1,7 for PbF_2, 1,42 for Y_2O_3 and 1,3 for YF_3. From these results YF_3 was chosen as low refractive index material in the coating designs. Multi-layer coatings incorporating ZnS, Ge and YF_3 films were designed and evaporated. Measured reflectance values below 0,2% were obtained from 9 μm to 11 mum. These systems were stable and robust. Finally, a silicon ball lens was

  13. Surprisingly high substrate specificities observed in complex biofilms

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Kindaichi, Tomonori; Kragelund, Caroline;

    to investigate the cell-specific in situ substrate uptake pattern of different bacteria. Different substrates were tested by combination of Microautoradiography and Fluorescence in situ Hybridization. Conditions applied (different substrate concentrations, starvation, induction with specific substrates, multiple......, microorganisms can regulate their metabolism expressing wide range of uptake and catabolic systems. However, ecophysiological studies of natural biofilms indicate that bacteria are very specialized in their choice of substrate, so even minor changes in substrate composition can affect the community composition...... by selection for different specialized species. We hypothesized that bacteria growing in natural environment express strongly conserved substrate specificity which is independent on short-term (few hours) variations in growth conditions. In this study, biofilm from Aalborg wastewater treatment plant was used...

  14. Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

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    Tyagi Sadhna

    2009-06-01

    Full Text Available Abstract Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand, although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

  15. Substrate specificity of the ubiquitin and Ubl proteases

    Science.gov (United States)

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  16. Crystal Structure and Substrate Specificity of PTPN12

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    Hui Li

    2016-05-01

    Full Text Available PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.

  17. Substrate Specificity and Structure of Human aminoadipate aminotransferase/kynurenine aminotransferase II

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Cai, T; Tagle, D; Robinson, H; Li, J

    2009-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  18. Substrate Specificity and Structure of Human Aminoadipate Aminotransferase/kynurenine Aminotransferase II

    Energy Technology Data Exchange (ETDEWEB)

    Han,Q.; Cai, T.; Tagle, D.; Robinson, H.; Li, J.

    2008-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to a-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested a-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with a-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  19. Characterizing Protease Specificity: How Many Substrates Do We Need?

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    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  20. Substrate Specificity of Peptide Adsorption: A Model Study

    OpenAIRE

    Bachmann, Michael; Janke, Wolfhard

    2007-01-01

    Applying the contact density chain-growth algorithm to lattice heteropolymers, we identify the conformational transitions of a nongrafted hydrophobic-polar heteropolymer with 103 residues in the vicinity of a polar, a hydrophobic, and a uniformly attractive substrate. Introducing only two system parameters, the numbers of surface contacts and intrinsic hydrophobic contacts, respectively, we obtain surprisingly complex temperature and solvent dependent, substrate-specific pseudo-phase diagrams.

  1. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

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    Tae-Kyun Oh

    2014-09-01

    Full Text Available A full-length phytase gene (phy of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR, and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5, an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F, the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  2. Force-driven aggregation of specific bonds on compliant substrates.

    Science.gov (United States)

    Sarvestani, Alireza S

    2013-07-26

    A thermodynamic model was employed to understand the underlying physics of the force-dependent size of cell's focal adhesions. The model describes the specific adhesion of an elastic membrane to a compliant substrate such that the adhesion site is subjected to the force of a constant pulling traction. The membrane contains mobile adhesion receptors with specific energetic affinities for complimentary ligands on the substrate. The adhesion is resisted by a disjoining pressure induced by a squeezed layer of glycocalyx. The model demonstrates that the enlargement of adhesion area with increasing pulling traction is possible due to a spontaneous response of the adhesion site to attain the state of minimum free energy. The correlation between the force and the adhesion area strongly depends on the rigidity of the substrate. PMID:23764177

  3. Molecular Cloning and Characterization of a Broad Substrate Terpenoid Oxidoreductase from Artemisia annua

    NARCIS (Netherlands)

    Ryden, Anna-Margareta; Ruyter-Spira, Carolien; Litjens, Ralph; Takahashi, Shunji; Quax, Wim; Osada, Hiroyuki; Bouwmeester, Harro; Kayser, Oliver

    2010-01-01

    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids, a

  4. Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.

    NARCIS (Netherlands)

    Ryden, A.M.; Ruyter-Spira, C.P.; Litjens, R.; Takahashi, S.; Quax, W.J.; Osada, H.; Bouwmeester, H.J.; Kayser, O.

    2010-01-01

    From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids,

  5. African Cultural and Literary Specificity in the Broad Translation Quality Debate

    OpenAIRE

    Wanchia T. Neba

    2015-01-01

    Beyond revisiting the byzantine and seemingly inconclusive debate on translation quality assurance and assessment, this article investigates the extent of an across-the-board applicability of existing quality assessment frameworks to the broad translation quality debate, against a strong backdrop of culture-specificity. It, first and foremost, exemplifies cultural and literary specificity through linguistically open-ended African creative writing, examines the variegated concept of translatio...

  6. A loop of coagulation factor VIIa influencing macromolecular substrate specificity

    DEFF Research Database (Denmark)

    Bjelke, Jais R; Persson, Egon; Rasmussen, Hanne B;

    2006-01-01

    . The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation....../Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation......Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa...

  7. Substrate specificity within a family of outer membrane carboxylate channels.

    Directory of Open Access Journals (Sweden)

    Elif Eren

    2012-01-01

    Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  8. Structural basis for substrate specificity of mammalian neuraminidases.

    Directory of Open Access Journals (Sweden)

    Victoria Smutova

    Full Text Available The removal of sialic acid (Sia residues from glycoconjugates in vertebrates is mediated by a family of neuraminidases (sialidases consisting of Neu1, Neu2, Neu3 and Neu4 enzymes. The enzymes play distinct physiological roles, but their ability to discriminate between the types of linkages connecting Sia and adjacent residues and between the identity and arrangement of the underlying sugars has never been systematically studied. Here we analyzed the specificity of neuraminidases by studying the kinetics of hydrolysis of BODIPY-labeled substrates containing common mammalian sialylated oligosaccharides: 3'Sia-LacNAc, 3'SiaLac, SiaLex, SiaLea, SiaLec, 6'SiaLac, and 6'SiaLacNAc. We found significant differences in substrate specificity of the enzymes towards the substrates containing α2,6-linked Sia, which were readily cleaved by Neu3 and Neu1 but not by Neu4 and Neu2. The presence of a branching 2-Fuc inhibited Neu2 and Neu4, but had almost no effect on Neu1 or Neu3. The nature of the sugar residue at the reducing end, either glucose (Glc or N-acetyl-D-glucosamine (GlcNAc had only a minor effect on all neuraminidases, whereas core structure (1,3 or 1,4 bond between D-galactose (Gal and GlcNAc was found to be important for Neu4 strongly preferring β3 (core 1 to β4 (core 2 isomer. Neu3 and Neu4 were in general more active than Neu1 and Neu2, likely due to their preference for hydrophobic substrates. Neu2 and Neu3 were examined by molecular dynamics to identify favorable substrate orientations in the binding sites and interpret the differences in their specificities. Finally, using knockout mouse models, we confirmed that the substrate specificities observed in vitro were recapitulated in enzymes found in mouse brain tissues. Our data for the first time provide evidence for the characteristic substrate preferences of neuraminidases and their ability to discriminate between distinct sialoside targets.

  9. Structural Basis of Substrate Specificity in Geobacter metallireducens SMUG1.

    Science.gov (United States)

    Zhang, Zhemin; Shen, Jiemin; Yang, Ye; Li, Jing; Cao, Weiguo; Xie, Wei

    2016-06-17

    Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are critical to maintain genome integrity, in which the base excision repair pathway plays an essential role. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for removing the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Geobacter metallireducens SMUG1 (GmeSMUG1) is an interesting family 3 enzyme in the UDG superfamily, with dual substrate specificities for DNA with uracil or xanthine. In contrast, the mutant G63P of GmeSMUG1 has exclusive activity for uracil, while N58D is inactive for both substrates, as we have reported previously. However, the structural bases for these substrate specificities are not well understood. In this study, we solved a series of crystal structures of WT and mutants of GmeSMUG1 at relatively high resolutions. These structures provide insight on the molecular mechanism of xanthine recognition for GmeSMUG1 and indicate that H210 plays a key role in xanthine recognition, which is in good agreement with the results of our EMSA and activity assays. More importantly, our mutant structures allow us to build models to rationalize our previous experimental observations of altered substrate activities of these mutants. PMID:27071000

  10. Molecular and Biochemical Characterization of a Novel β-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis

    Directory of Open Access Journals (Sweden)

    Fengyi Liu, Tian Liu, Mingbo Qu, Qing Yang

    2012-01-01

    Full Text Available Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B. These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa, and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.

  11. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  12. Molecular mechanism of strict substrate specificity of an extradiol dioxygenase, DesB, derived from Sphingobium sp. SYK-6.

    Directory of Open Access Journals (Sweden)

    Keisuke Sugimoto

    Full Text Available DesB, which is derived from Sphingobium sp. SYK-6, is a type II extradiol dioxygenase that catalyzes a ring opening reaction of gallate. While typical extradiol dioxygenases show broad substrate specificity, DesB has strict substrate specificity for gallate. The substrate specificity of DesB seems to be required for the efficient growth of S. sp. SYK-6 using lignin-derived aromatic compounds. Since direct coordination of hydroxyl groups of the substrate to the non-heme iron in the active site is a critical step for the catalytic reaction of the extradiol dioxygenases, the mechanism of the substrate recognition and coordination of DesB was analyzed by biochemical and crystallographic methods. Our study demonstrated that the direct coordination between the non-heme iron and hydroxyl groups of the substrate requires a large shift of the Fe (II ion in the active site. Mutational analysis revealed that His124 and His192 in the active site are essential to the catalytic reaction of DesB. His124, which interacts with OH (4 of the bound gallate, seems to contribute to proper positioning of the substrate in the active site. His192, which is located close to OH (3 of the gallate, is likely to serve as the catalytic base. Glu377' interacts with OH (5 of the gallate and seems to play a critical role in the substrate specificity. Our biochemical and structural study showed the substrate recognition and catalytic mechanisms of DesB.

  13. Tocopherol Cyclases—Substrate Specificity and Phylogenetic Relations

    Science.gov (United States)

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B.; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

  14. Substrate specificity of allelic variants of the TAP peptide transporter.

    Science.gov (United States)

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant.

  15. Substrate specificity of allelic variants of the TAP peptide transporter.

    Science.gov (United States)

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant. PMID:7895166

  16. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  17. Highly efficient broad-area blue and white light-emitting diodes on bulk GaN substrates

    Energy Technology Data Exchange (ETDEWEB)

    Vampola, Kenneth J.; Fellows, Natalie N.; Masui, Hisashi; Chung, Roy B.; Sato, Hitoshi; Sonoda, Junichi; Hirasawa, Hirohiko; Iza, Michael; Nakamura, Shuji [Materials Department, University of California, Santa Barbara, California (United States); Brinkley, Stuart E.; Furukawa, Motoko [Electrical and Computer Engineering Department, University of California, Santa Barbara, California (United States); DenBaars, Steven P. [Materials Department, University of California, Santa Barbara, California (United States)]|[Electrical and Computer Engineering Department, University of California, Santa Barbara, California (United States)

    2009-02-15

    Highly efficient light emitting diodes (LEDs) with peak emission wavelengths of nominally 450 nm were grown, fabricated and tested. The growth was performed by metal organic chemical vapour deposition. The LEDs were grown on c-plane (0001) bulk GaN substrates and fabricated into broad-area devices with active area 0.01 cm{sup 2}. Considerations were made to improve extraction efficiency, including transparent contacts, suspended mirror-less packaging and encapsulation in a truncated pyramid optic. These factors resulted in LEDs with high peak external quantum efficiency and reduced efficiency droop. The output power and external quantum efficiency at 20 mA were 38.5 mW and 68.9%. At 100 mA, they were 170 mW and 60.9%. White LEDs were fabricated by application of a yellow phosphor to the blue LEDs. The white LED luminous flux and efficacy at 20 mA was 9.6 lm and 128 lm/W. The chromaticity coordinates and correlated colour temperature were (0.348 K, 0.378 K) and 4998 K. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  18. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng

    2008-01-01

    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  19. An ABC-type multidrug transporter of Lactococcus lactis possesses an exceptionally broad substrate specificity

    NARCIS (Netherlands)

    Poelarends, GJ; Mazurkiewicz, P; Putman, M; Cool, RH; van Veen, HW; Konings, WN

    2000-01-01

    LmrA is a 590-amino acid membrane protein which confers multidrug resistance on Lactococcus lactis cells by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane at the expense of ATP hydrolysis. Its structural and functional characteristics place it in the P-glycoprotei

  20. Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in Vitro.

    Science.gov (United States)

    Shi, Wan; Zeng, Qin; Kunkel, Barbara N; Running, Mark P

    2016-01-15

    Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double-geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1·RGTB1 and RGTA1·RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can not only prenylate a great variety of Rab GTPases in the presence of Rab escort protein but, unlike Rab-GGT in yeast and mammals, can also prenylate certain non-Rab GTPases independently of Rab escort protein. Our findings may help to explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants. PMID:26589801

  1. Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1.

    Science.gov (United States)

    Dewitte, Griet; Walmagh, Maarten; Diricks, Margo; Lepak, Alexander; Gutmann, Alexander; Nidetzky, Bernd; Desmet, Tom

    2016-09-10

    UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource.

  2. Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1.

    Science.gov (United States)

    Dewitte, Griet; Walmagh, Maarten; Diricks, Margo; Lepak, Alexander; Gutmann, Alexander; Nidetzky, Bernd; Desmet, Tom

    2016-09-10

    UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource. PMID:27378621

  3. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    Science.gov (United States)

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments. PMID:26672451

  4. Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors.

    Science.gov (United States)

    Franek, Milan; Diblikova, Iva; Cernoch, Ivo; Vass, Maria; Hruska, Karel

    2006-03-01

    Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.

  5. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

    NARCIS (Netherlands)

    Kamerbeek, NM; Olsthoorn, AJJ; Fraaije, MW; Janssen, DB; Kamerbeek, Nanne M.; Olsthoorn, Arjen J.J.

    2003-01-01

    The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of ca

  6. Substrate specificity of papain dynamic structures for peptides consisting of 8-10 GLY residues

    Science.gov (United States)

    Nishiyama, Katsuhiko

    2011-01-01

    We investigated the substrate specificity of papain dynamic structures for peptides of 8-10 glycine residues (8-10GLY) via molecular dynamics and docking simulations. The substrate specificity of papain for 8-10GLY fluctuated considerably with time. There were several residues that were different among those that had a significant impact on binding (RESIDUES_IMPACT) with 10GLY, 9GLY, and 8GLY. Modification of these different residues should allow for control of substrate specificity, providing a framework for modifying substrate specificity in papain and other enzymes.

  7. Broadly tuned, view-specific coding of face shape: opposing figural aftereffects can be induced in different views.

    Science.gov (United States)

    Jeffery, Linda; Rhodes, Gillian; Busey, Tom

    2007-11-01

    Face aftereffects are sensitive to changes in viewpoint, suggesting view-specific face coding, yet are not entirely eliminated by changes in viewpoint, suggesting view-invariance. To determine whether broad view-tuning can account for these findings we measured the reduction of a figural face aftereffect induced in one view by concurrent adaptation to an opposite distortion in a second viewpoint, varying the angle between these views. To the degree that the same neural population codes both viewpoints, the opposing aftereffects should cancel. Cancellation increased monotonically as the angle between two adapting views decreased, consistent with broadly tuned, view-specific coding of face shape. PMID:17920099

  8. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    Science.gov (United States)

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

  9. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

    Directory of Open Access Journals (Sweden)

    Chaikuad Apirat

    2012-06-01

    Full Text Available Abstract Backround Aspartyl aminopeptidase (DNPEP, with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

  10. Lipid substrate specificity of phosphatidylethanolamine N-methyltransferase of Tetrahymena

    International Nuclear Information System (INIS)

    The ciliate protozoan Tetrahymena thermophila forms about 60% of its phosphatidylcholine by methylation of phosphatidylethanolamine with S-adenosylmethionine using the enzyme phosphatidylethanolamine N-methyltransferase. Analogues of ethanolamine or of ethanolamine phosphate are incorporated into the phospholipids of Tetrahymena when cells are cultured in their presence. These compounds, 3-amino-1-propanol, 2-aminoethylphosphonate, 3-aminopropylphosphonate and N,N-dimethylaminoethylphosphonate replace from 50 to 75% of the ethanolamine phosphate in phosphatidylethanolamine. However, analysis of the phospholipids of lipid-altered Tetrahymena showed that none of the phosphatidylethanolamine analogues had been converted to the corresponding phosphatidylcholine analogue. No incorration of [14C-CH3]methionine into the phosphatidylcholine analogues could be demonstrated in vivo, nor was label from [3H-CH3]S-adenosylmethionine incorporated in virto. Thus, only phosphatidylethanolamine and its monomethyl and dimethyl derivatives have been found to be substrates for the phosphatidylethanoiamine N-methyltransferase. The enzyme therefore requires a phospholipid substrate containing an ester linkage between the alkylamine and phosphorus, with the amino group required to be β to the alcohol

  11. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  12. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    Directory of Open Access Journals (Sweden)

    Woo-Suk Jung

    Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  13. A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis†

    OpenAIRE

    Liu, Ce Feng; Liu, Dali; Momb, Jessica; Thomas, Pei W.; Lajoie, Ashley; Petsko, Gregory A; Fast, Walter; Ringe, Dagmar

    2013-01-01

    AiiA is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center, but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the prese...

  14. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    OpenAIRE

    Sheng-Bao Suo; Jian-Ding Qiu; Shao-Ping Shi; Xiang Chen; Ru-Ping Liang

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by...

  15. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

    Energy Technology Data Exchange (ETDEWEB)

    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat (UNM); (IIT); (UMBI)

    2008-04-02

    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  16. Myxozoan infections of caecilians demonstrate broad host specificity and indicate a link with human activity.

    Science.gov (United States)

    Hartigan, Ashlie; Wilkinson, Mark; Gower, David J; Streicher, Jeffrey W; Holzer, Astrid S; Okamura, Beth

    2016-05-01

    Myxozoans are parasitic cnidarians that infect a wide variety of hosts. Vertebrates typically serve as intermediate hosts whereas definitive hosts are invertebrates, including annelids and bryozoans. Myxozoans are known to exploit species in two of the three extant amphibian orders (Anura: frogs and toads; Caudata: newts and salamanders). Here we use museum collections to determine, to our knowledge for the first time, whether myxozoans also exploit the third amphibian order (Gymnophiona: caecilians). Caecilians are a poorly known group of limbless amphibians, the ecologies of which range from aquatic to fully terrestrial. We examined 12 caecilian species in seven families (148 individuals total) characterised by a diversity of ecologies and life histories. Using morphological and molecular surveys, we discovered the presence of the myxozoan Cystodiscus axonis in two South American species (one of seven examined families) of aquatic caecilians - Typhlonectes natans and Typhlonectes compressicauda. All infected caecilians had been maintained in captivity in the United Kingdom prior to their preservation. Cystodiscus axonis is known from several Australian frog species and its presence in caecilians indicates a capacity for infecting highly divergent amphibian hosts. This first known report of myxozoan infections in caecilians provides evidence of a broad geographic and host range. However, the source of these infections remains unknown and could be related to exposure in South America, the U.K. or to conditions in captivity. PMID:26945641

  17. Engineering Substrate Preference in Subtilisin: Structural and Kinetic Analysis of a Specificity Mutant

    Energy Technology Data Exchange (ETDEWEB)

    Ruan, Biao; London, Viktoriya; Fisher, Kathryn E.; Gallagher, D. Travis; Bryan, Philip N. (UMBI)

    2008-08-06

    Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k{sub cat} for the reaction. This also leads to strong product inhibition of substrate

  18. Amino acid residues important for substrate specificity of the amino acid permeases Can I p and Gnp I p in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Kielland-Brandt, M.C.

    2001-01-01

    in the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[C-14]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[C-14......]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations...

  19. Long-term nonprogression and broad HIV-1-specific proliferative T-cell responses

    Directory of Open Access Journals (Sweden)

    Nesrina eImami

    2013-03-01

    Full Text Available Complex mechanisms underlying the maintenance of fully functional, proliferative, HIV-1-specific T-cell responses involve processes from early T-cell development through to the final stages of T-cell differentiation and antigen recognition. Virus-specific proliferative CD4 and CD8 T-cell responses, important for the control of infection, are observed in some HIV-1+ patients during early stages of disease, and are maintained in long-term nonprogressing subjects. In the vast majority of HIV-1+ patients, full immune functionality is lost when proliferative HIV-1-specific T-cell responses undergo a variable progressive decline throughout the course of chronic infection. This appears irreparable despite administration of potent combination antiretroviral therapy, which to date is non-curative, necessitating life-long administration and the development of effective, novel, therapeutic interventions. While a sterilising cure, involving clearance of virus from the host, remains a primary aim, a functional cure may be a more feasible goal with considerable impact on worldwide HIV-1 infection. Such an approach would enable long-term co-existence of host and virus in the absence of toxic and costly drugs. Effective immune homeostasis coupled with a balanced response appropriately targeting conserved viral antigens, in a manner that avoids hyperactivation and exhaustion, may prove to be the strongest correlate of durable viral control. This review describes novel concepts underlying full immune functionality in the context of HIV-1 infection, which may be utilised in future strategies designed to improve upon existing therapy. The aim will be to induce long-term nonprogressor or elite controller status in every infected host, through immune-mediated control of viraemia and reduction of viral reservoirs, leading to lower HIV-1 transmission rates.

  20. The functional consequences of relative substrate specificity in complex biochemical systems

    Directory of Open Access Journals (Sweden)

    Yan eZeng

    2011-09-01

    Full Text Available A biochemical activity, that is, enzymatic reaction or molecular interaction, frequently involves a molecule, for example, an enzyme, capable of interacting with numerous substrates or partners. Specificity is a fundamental property of biochemical activities, and relative specificity refers to the situation whereby a molecule interacts with multiple substrates or partners but with different affinities. Here, a hypothesis is proposed that any molecule, such as an enzyme, would have a range of preferences or relative specificity for its many native substrates, which differentially impacts the phenotypes of these substrates and hence shapes the relevant biological processes in vivo. While the mechanisms underlying the specific recognition between enzymes and individual substrates have been studied extensively, whether any enzyme exhibits intrinsic selectivity towards its ensemble of substrates is often overlooked, and whether this selectivity has any functional consequences is much less appreciated. There are, however, several lines of evidence in the literature that are consistent with the hypothesis and reviewed here. Furthermore, this hypothesis is supported by our analyses of a number of diverse biochemical systems at a large scale. Thus, the human microRNA processing machinery possesses relative specificity towards its hundreds of substrates, which might contribute to differential microRNA biogenesis; the promoter binding affinity of the transcription factor Ndt80 might regulate Ndt80 target mRNA expression in the budding yeast; Cdk1 kinase specificity might lead to variable substrate phosphorylation in vivo; and the density of HuR deposition to its thousands of RNA targets might partly explain differential RNA expression in human cells. It is proposed, therefore, that relative specificity is a universal property of complex biochemical systems and that the hypothesis could denote a general principle in biology.

  1. Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

    Directory of Open Access Journals (Sweden)

    Nitish K Mishra

    Full Text Available BACKGROUND: Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task. RESULTS: Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset. CONCLUSIONS: Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models

  2. Tissue-specific and substrate-specific mitochondrial bioenergetics in feline cardiac and skeletal muscles

    DEFF Research Database (Denmark)

    Christiansen, Liselotte Bruun; Dela, Flemming; Koch, Jørgen;

    2015-01-01

    fibers. Biopsies of left ventricular cardiac muscle and soleus muscle, a type I-rich oxidative skeletal muscle, were obtained from 15 healthy domestic cats. Enzymatic activity of citrate synthase (CS), a biomarker of mitochondrial content, was measured. Mitochondrial OXPHOS capacity with various kinds...... of non-fatty-acid substrates and fatty-acid substrate in permeabilized muscle fiber was measured by using high-resolution respirometry. CS activity in the heart was 3 times higher than in the soleus muscle. Mitochondrial state 3 respiration, ADP-stimulated respiration, with complex I-linked and complex I...

  3. Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant meso-diaminopimelate dehydrogenase.

    Science.gov (United States)

    Liu, Weidong; Guo, Rey-Ting; Chen, Xi; Li, Zhe; Gao, Xiuzhen; Huang, Chun-Hsiang; Wu, Qiaqing; Feng, Jinhui; Zhu, Dunming

    2015-04-13

    A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases. PMID:25754803

  4. Study on Substrate Specificity at Subsites for Severe Acute Respiratory Syndrome Coronavirus 3CL Protease

    Institute of Scientific and Technical Information of China (English)

    Yu-Fei SHAN; Gen-Jun XU

    2005-01-01

    Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2'. The deletion of either P4 or P2' residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S 1' subsite, we have found that bulky residues such as Tyr and Trp were also acceptable.In addition, based on both peptide-based assay and autocleavage assay, Ile at the P1' position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S 1' subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2' position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.

  5. Characterization of HIV-Specific CD4+T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

    DEFF Research Database (Denmark)

    Buggert, Marcus; Norstrom, Melissa M.; Czarnecki, Chris;

    2012-01-01

    for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve...... this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73...... II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines....

  6. Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

    Directory of Open Access Journals (Sweden)

    Huimin Liu

    2014-06-01

    Full Text Available Cysteine protease 1 precursor from Zea mays (zmCP1 is classified as a member of the C1A family of peptidases (papain-like cysteine protease in MEROPS (the Peptidase Database. The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe. Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.

  7. [Substrate specificity of carotenoid 3',4'-desaturase from Deinococcus radiodurans].

    Science.gov (United States)

    Sun, Zongtao; Tian, Bing; Shen, Shaochuan; Hu, Yuejin

    2010-10-01

    To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBI(Eu) and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').

  8. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms.

    Science.gov (United States)

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta

    2016-01-01

    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms. PMID:27576440

  9. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

    Science.gov (United States)

    McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas

    2016-01-01

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype. PMID:26907590

  10. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.

    2013-06-06

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  11. Quantitation of HIV-1 DNA with a sensitive TaqMan assay that has broad subtype specificity.

    Science.gov (United States)

    van der Sluis, Renée M; van Montfort, Thijs; Centlivre, Mireille; Schopman, Nick C T; Cornelissen, Marion; Sanders, Rogier W; Berkhout, Ben; Jeeninga, Rienk E; Paxton, William A; Pollakis, Georgios

    2013-01-01

    The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form. PMID:23059551

  12. Site specific isolated nanostructure array formation on a large area by broad ion beam without any mask and resist

    Science.gov (United States)

    Karmakar, Prasanta; Satpati, Biswarup

    2014-06-01

    We report the formation of isolated nanostructure arrays on a large area via broad ion beam implantation without the aid of any mask or resist. Desired ions have been implanted at specific locations of the prefabricated silicon ripple or triangular structures by exploiting the variation of local ion impact angles. We have shown that the implantation of Fe ions on an O+ ions induced pre fabricated triangular shaped patterned Si surface results in a self-organized periodic array of striped magnetic nanostructures having several micron length and about 50 nm width arranged with a spacial separation of ˜200 nm. The morphology, composition, crystalline structure, and magnetic property of these nanopatterns have been analyzed using high-resolution cross-sectional transmission electron microscopy and atomic force microscopy. A geometrical model has been proposed to explain the fundamental features of such ion-induced nanopattern structures.

  13. Site specific isolated nanostructure array formation on a large area by broad ion beam without any mask and resist

    International Nuclear Information System (INIS)

    We report the formation of isolated nanostructure arrays on a large area via broad ion beam implantation without the aid of any mask or resist. Desired ions have been implanted at specific locations of the prefabricated silicon ripple or triangular structures by exploiting the variation of local ion impact angles. We have shown that the implantation of Fe ions on an O+ ions induced pre fabricated triangular shaped patterned Si surface results in a self-organized periodic array of striped magnetic nanostructures having several micron length and about 50 nm width arranged with a spacial separation of ∼200 nm. The morphology, composition, crystalline structure, and magnetic property of these nanopatterns have been analyzed using high-resolution cross-sectional transmission electron microscopy and atomic force microscopy. A geometrical model has been proposed to explain the fundamental features of such ion-induced nanopattern structures.

  14. Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool.

    Science.gov (United States)

    Sannerud, Ragna; Esselens, Cary; Ejsmont, Paulina; Mattera, Rafael; Rochin, Leila; Tharkeshwar, Arun Kumar; De Baets, Greet; De Wever, Veerle; Habets, Roger; Baert, Veerle; Vermeire, Wendy; Michiels, Christine; Groot, Arjan J; Wouters, Rosanne; Dillen, Katleen; Vints, Katlijn; Baatsen, Pieter; Munck, Sebastian; Derua, Rita; Waelkens, Etienne; Basi, Guriqbal S; Mercken, Mark; Vooijs, Marc; Bollen, Mathieu; Schymkowitz, Joost; Rousseau, Frederic; Bonifacino, Juan S; Van Niel, Guillaume; De Strooper, Bart; Annaert, Wim

    2016-06-30

    γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis. PMID:27293189

  15. Computational approaches for classification and prediction of P-type ATPase substrate specificity in Arabidopsis.

    Science.gov (United States)

    Zinati, Zahra; Alemzadeh, Abbas; KayvanJoo, Amir Hossein

    2016-01-01

    As an extended gamut of integral membrane (extrinsic) proteins, and based on their transporting specificities, P-type ATPases include five subfamilies in Arabidopsis, inter alia, P4ATPases (phospholipid-transporting ATPase), P3AATPases (plasma membrane H(+) pumps), P2A and P2BATPases (Ca(2+) pumps) and P1B ATPases (heavy metal pumps). Although, many different computational methods have been developed to predict substrate specificity of unknown proteins, further investigation needs to improve the efficiency and performance of the predicators. In this study, various attribute weighting and supervised clustering algorithms were employed to identify the main amino acid composition attributes, which can influence the substrate specificity of ATPase pumps, classify protein pumps and predict the substrate specificity of uncharacterized ATPase pumps. The results of this study indicate that both non-reduced coefficients pertaining to absorption and Cys extinction within 280 nm, the frequencies of hydrogen, Ala, Val, carbon, hydrophilic residues, the counts of Val, Asn, Ser, Arg, Phe, Tyr, hydrophilic residues, Phe-Phe, Ala-Ile, Phe-Leu, Val-Ala and length are specified as the most important amino acid attributes through applying the whole attribute weighting models. Here, learning algorithms engineered in a predictive machine (Naive Bays) is proposed to foresee the Q9LVV1 and O22180 substrate specificities (P-type ATPase like proteins) with 100 % prediction confidence. For the first time, our analysis demonstrated promising application of bioinformatics algorithms in classifying ATPases pumps. Moreover, we suggest the predictive systems that can assist towards the prediction of the substrate specificity of any new ATPase pumps with the maximum possible prediction confidence. PMID:27186030

  16. Most substrates suitable if you adapt the watering and fertiliser : take note of specific plants needs

    NARCIS (Netherlands)

    Heuvelink, E.; Kierkels, T.

    2012-01-01

    The diversity in substrates keeps growing. You can't say that one is better than the other if you take into account their characteristics and adapt the watering and fertilisation. But you also need take into account the specific requirements of the plant which we'll discuss in this article.

  17. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.

    Science.gov (United States)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram; Madsen, Thomas Daugbjerg; Gerken, Thomas A; Vester-Christensen, Malene B; Wandall, Hans H; Bennett, Eric Paul; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

  18. Substrate Specificity of Cyclic Nucleotide Phosphodiesterase from Beef Heart and from Dictyostelircm discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Dijkgraaf, Peter A.M.; Konijn, Theo M.; Abbad, Emilio Garcia; Petridis, Georg; Jastorff, Bernd

    1983-01-01

    The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3’,5’-monophosph

  19. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anke K Trilling

    Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

  20. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds

    Science.gov (United States)

    Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to...

  1. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    International Nuclear Information System (INIS)

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases

  2. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    Science.gov (United States)

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  3. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

    DEFF Research Database (Denmark)

    Larsen, Katrine S; Østergaard, Henrik; Bjelke, Jais R;

    2007-01-01

    of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather...... promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen...

  4. Specific Heat of Hollow Nanosphere Coupled to Substrate: Quantum Size Effects

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    With the help of the elastic wave theory, in the perturbed approximation the density-of-states for vibrational modes and the specific heat axe studied for different hollow Si nanospheres, coupled with a semi-infinite substrate. We find that the modes of such coupled hollow spheres are significantly broadened and shifted toward low frequencies. The specific heat of the coupled hollow nanosphere is bigger than an isolated one due to the coupling interaction and quantum size effects. The predicted coupling and size enhancements on specific heat are probed in thermal experiments.

  5. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    Science.gov (United States)

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  6. Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus.

    Science.gov (United States)

    Lukacin, Richard; Matern, Ulrich; Specker, Silvia; Vogt, Thomas

    2004-11-19

    Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

  7. New chemiluminescent substrates of paraoxonase 1 with improved specificity: synthesis and properties.

    Science.gov (United States)

    Abulimite, Zulipiyan; Mu, Xiaojing; Xiao, Shangyou; Liu, Min; Li, Quandan; Chen, Gang

    2015-05-01

    Paraoxonase 1 (PON1) is an important hydrolase, and the enzyme activity decreases in patients with liver disease, diabetes, coronary heart disease, etc. Phenyl acetate and organophosphates are usually employed as substrates for serum PON1 activity assay. However, phenyl acetate for arylesterase activity assay exhibits disadvantage of high background. According to properties of PON1, four new chemiluminescent acridinium esters were designed, prepared through three steps, and characterized with (1)H NMR and mass spectrometry (MS) data, and their properties as PON1 substrates were investigated. The hydrolyses of the four compounds catalyzed by recombinant human PON1 (rhPON1) (or serum) followed first-order kinetics within 22 min. The PON1 activator (NaCl, 0.10 mol L(-1)) could boost the rhPON1-mediated and serum-mediated hydrolyses of the acridinium esters to 2.01 ~ 2.26 folds, but 1.0 mol L(-1) NaCl decreased the serum arylesterase activity. RhPON1 showed selectivity over other serum esterases such as lipase, acetylcholinesterase, and esterase D more than 300 folds. By using ethylene diamine tetraacetic acid (EDTA) inhibitor, the specificities of the four substrates toward serum PON1 were determined as 78.3 ~ 92.9%, which is improved than that of the model compound 9-(4-chloro-phenoxycarbonyl)-10-methylacridinium ester triflate. Due to low toxicity, high specificity, and sensitivity of the substrates, they are useful for serum PON1 activity assay. PMID:25809994

  8. Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

    Science.gov (United States)

    Kalupov, Timofey; Brillard-Bourdet, Michèle; Dadé, Sébastien; Serrano, Hélène; Wartelle, Julien; Guyot, Nicolas; Juliano, Luiz; Moreau, Thierry; Belaaouaj, Azzaq; Gauthier, Francis

    2009-01-01

    It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases. PMID:19833730

  9. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank

    2010-09-01

    Full Text Available Peroxidases (POD and polyphenol oxidase (PPO are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

  10. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    Science.gov (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  11. Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity.

    Science.gov (United States)

    Fallgren, C; Utt, M; Ljungh, A

    2001-06-01

    A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins. PMID:11393292

  12. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve.

    Science.gov (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O; Marasco, Wayne A

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of 'universal' influenza vaccine strategies. PMID:27619409

  13. Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

    NARCIS (Netherlands)

    Kale, Avinash; Pijning, Tjaard; Sonke, Theo; Dijkstra, Bauke W.; Thunnissen, Andy-Mark W. H.; Guss, M.

    2010-01-01

    The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure function relationships, the enzyme was studied by X-ray crystallography. Crystal str

  14. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    Science.gov (United States)

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism. PMID:23800623

  15. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases.

    Science.gov (United States)

    Waldner, Birgit J; Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-01-21

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design.

  16. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

    Science.gov (United States)

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans

    2007-03-01

    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  17. The substrate specificities of sunflower and soybean phospholipases D using transphosphatidylation reaction

    Directory of Open Access Journals (Sweden)

    Abdelkafi Slim

    2011-11-01

    Full Text Available Abstract Background Phospholipase D (PLD belongs to a lipolytic enzyme subclass which catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond. Results In this work, we have studied the substrate specificity of PLDs from germinating sunflower seeds and cultured-soybean cells, using their capacity of transphosphatidylation. In the presence of a nucleophilic acceptor, such as [14C]ethanol, PLD catalyzes the production of phosphatidyl-[14C]-ethanol. The resulting product is easily identified since it is well separated from the other lipids by thin-layer chromatography. The main advantage of this assay is that the phospholipid used as substrate does not need to be radiolabelled and thus allow us a large choice of polar heads and fatty acids. In vitro, we observed that sunflower and soybean cell PLD show the following decreasing order of specificity: phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol; while phosphatidylserine and phosphatidylinositol are utilized much less efficiently. Conclusions The substrate specificity is modulated by the fatty acid composition of the phosphatidylcholine used as well as by the presence of other charged phospholipids.

  18. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J

    2010-01-01

    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  19. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    Science.gov (United States)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  20. Crystal structure and substrate specificity of Drosophila 3,4-dihydroxyphenylalanine decarboxylase.

    Directory of Open Access Journals (Sweden)

    Qian Han

    Full Text Available BACKGROUND: 3,4-Dihydroxyphenylalanine decarboxylase (DDC, also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. PRINCIPAL FINDINGS: In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. CONCLUSIONS: The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  1. Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities.

    Science.gov (United States)

    Sommer, Bettina; Overy, David P; Haltli, Bradley; Kerr, Russell G

    2016-07-01

    Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa. PMID:27130210

  2. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    Full Text Available Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

  3. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    Science.gov (United States)

    Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design. PMID:26496636

  4. Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in hyper-arid environments

    Directory of Open Access Journals (Sweden)

    Alexander eCrits-Christoph

    2016-03-01

    Full Text Available Under extreme water deficit, endolithic (inside rock microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP and polyketides (PK. Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both

  5. Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments.

    Science.gov (United States)

    Crits-Christoph, Alexander; Robinson, Courtney K; Ma, Bing; Ravel, Jacques; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; Souza-Egipsy, Virginia; Casero, M Cristina; DiRuggiero, Jocelyne

    2016-01-01

    Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates

  6. Using bacteria to determine protein kinase specificity and predict target substrates.

    Directory of Open Access Journals (Sweden)

    Michael F Chou

    Full Text Available The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A and acidophilic (Casein Kinase II kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

  7. Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

    Science.gov (United States)

    Osuji, Godson O; Braithwaite, Cleantis; Fordjour, Kusi; Madu, Wenceslaus C; Beyene, Asefa; Roberts, Paul S; Wright, Victor

    2003-02-01

    Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

  8. The unique substrate specificity of human AOC2, a semicarbazide-sensitive amine oxidase.

    Science.gov (United States)

    Kaitaniemi, Sam; Elovaara, Heli; Grön, Kirsi; Kidron, Heidi; Liukkonen, Janne; Salminen, Tiina; Salmi, Marko; Jalkanen, Sirpa; Elima, Kati

    2009-08-01

    Semicarbazide-sensitive amine oxidases (SSAOs) catalyze oxidative deamination of primary amines, but the true physiological function of these enzymes is still poorly understood. Here, we have studied the functional and structural characteristics of a human cell-surface SSAO, AOC2, which is homologous to the better characterized family member, AOC3. The preferred in vitro substrates of AOC2 were found to be 2-phenylethylamine, tryptamine and p-tyramine instead of methylamine and benzylamine, the favored substrates of AOC3. Molecular modeling suggested structural differences between AOC2 and AOC3, which provide AOC2 with the capability to use the larger monoamines as substrates. Even though AOC2 mRNA was expressed in many tissues, the only tissues with detectable AOC2-like enzyme activity were found in the eye. Characterization of AOC2 will help in evaluating the contribution of this enzyme to the pathological processes attributed to the SSAO activity and in designing specific inhibitors for the individual members of the SSAO family. PMID:19588076

  9. An in vitro assay to study the recruitment and substrate specificity of chromatin modifying enzymes

    Directory of Open Access Journals (Sweden)

    Vermeulen Michiel

    2004-01-01

    Full Text Available Post-translational modifications of core histones play an important role in regulating fundamental biological processes such as DNA repair, transcription and replication. In this paper, we describe a novel assay that allows sequential targeting of distinct histone modifying enzymes to immobilized nucleosomal templates using recombinant chimeric targeting molecules. The assay can be used to study the histone substrate specificity of chromatin modifying enzymes as well as whether and how certain enzymes affect each other's histone modifying activities. As such the assay can help to understand how a certain histone code is established and interpreted.

  10. Dynamic changes in the secondary structure of ECE-1 and XCE account for their different substrate specificities

    Directory of Open Access Journals (Sweden)

    Ul-Haq Zaheer

    2012-11-01

    Full Text Available Abstract Background X-converting enzyme (XCE involved in nervous control of respiration, is a member of the M13 family of zinc peptidases, for which no natural substrate has been identified yet. In contrast, it’s well characterized homologue endothelin-converting enzyme-1 (ECE-1 showed broad substrate specificity and acts as endopeptidase as well as dipeptidase. To explore the structural differences between XCE and ECE-1, homology model of XCE was built using the complex structure of ECE-1 with phosphoramidon (pdb-id: 3DWB as template. Phosphoramidon was docked into the binding site of XCE whereas phosphate oxygen of the inhibitor was used as water molecule to design the apo forms of both enzymes. Molecular dynamics simulation of both enzymes was performed to analyze the dynamic nature of their active site residues in the absence and presence of the inhibitor. Results Homology model of XCE explained the role of non-conserved residues of its S2’ subsite. Molecular dynamics (MD simulations identified the flexible transitions of F149/I150, N566/N571, W714/W719, and R145/R723 residues of ECE-1/XCE for the strong binding of the inhibitor. Secondary structure calculations using DSSP method reveals the folding of R145/R723 residue of ECE-1/XCE into β-sheet structure while unfolding of the S2’ subsite residues in aECE-1 and sustained compact folding of that of aXCE. The results evaluated are in good agreement with available experimental data, thus providing detailed molecular models which can explain the structural and specificities differences between both zinc peptidases. Conclusions Secondary structure changes of both enzymes during the simulation time revealed the importance of β-sheet structure of R145/R723 for its binding with the terminal carboxylate group of the inhibitor. Unfolding of the α-helix comprising the S2’ subsite residues in aECE-1 correlate well with its endopeptidase activity while their compact folding in aXCE may

  11. The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II.

    Science.gov (United States)

    Sivaraman, Sharada; Kirsch, Jack F

    2006-05-01

    Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

  12. Characterization of substrate specificity of a rice silicon transporter, Lsi1.

    Science.gov (United States)

    Mitani, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2008-07-01

    Lsi1 (OsNIP2;1) is the first silicon (silicic acid) transporter identified in plant, which belongs to the nodulin 26-like intrinsic membrane protein (NIP) subfamily. In this study, we characterized the function of this transporter by using the Xenopus laevis oocyte expression system. The transport activity of Lsi1 for silicic acid was significantly inhibited by HgCl2 but not by low temperature. Lsi1 also showed an efflux transport activity for silicic acid. The substrate specificity study showed that Lsi1 was able to transport urea and boric acid; however, the transport activity for silicic acid was not affected by the presence of equimolar urea and was decreased only slightly by boric acid. Furthermore, among the NIPs subgroup, OsNIP2;2 showed transport activity for silicic acid, whereas OsNIP1;1 and OsNIP3;1 did not. We propose that Lsi1 and its close homologues form a unique subgroup of NIP with a distinct ar/R selectivity filter, which is located in the narrowest region on the extra-membrane mouth and govern the substrate specificity of the pore. PMID:18214526

  13. High-throughput profiling of N-myristoylation substrate specificity across species including pathogens.

    Science.gov (United States)

    Traverso, José A; Giglione, Carmela; Meinnel, Thierry

    2013-01-01

    One of the most critical modifications affecting the N-terminus of proteins is N-myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-myristoylation specificity. We show that myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.

  14. Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities.

    Science.gov (United States)

    Fesko, Kateryna

    2016-03-01

    Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable β-hydroxy-α-amino acids with complete stereocontrol at the α-carbon and moderate specificity at the β-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies have been conducted to improve the reaction protocols to enable higher conversions and investigate the substrate scope of enzymes. However, the application of threonine aldolases in organic synthesis is still limited due to often moderate yields and low diastereoselectivities obtained in the aldol reaction. This review briefly summarizes the screening techniques recently applied to discover novel threonine aldolases as well as enzyme engineering and mutagenesis studies which were accomplished to improve the catalytic activity and substrate specificity. Additionally, the results from new investigations on threonine aldolases including crystal structure determinations and structural-functional characterization are reviewed. PMID:26810201

  15. Carotenoids in Rhodoplanes species: variation of compositions and substrate specificity of predicted carotenogenesis enzymes.

    Science.gov (United States)

    Takaichi, Shinichi; Sasikala, Ch; Ramana, Ch V; Okamura, Keiko; Hiraishi, Akira

    2012-08-01

    Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

  16. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    Science.gov (United States)

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  17. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

    DEFF Research Database (Denmark)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram;

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr...... and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual...... isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide...

  18. Global, Broad, or Specific Cognitive Differences? Using a MIMIC Model to Examine Differences in CHC Abilities in Children with Learning Disabilities

    Science.gov (United States)

    Niileksela, Christopher R.; Reynolds, Matthew R.

    2014-01-01

    This study was designed to better understand the relations between learning disabilities and different levels of latent cognitive abilities, including general intelligence (g), broad cognitive abilities, and specific abilities based on the Cattell-Horn-Carroll theory of intelligence (CHC theory). Data from the "Differential Ability…

  19. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues

    Science.gov (United States)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

  20. Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

    Science.gov (United States)

    A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

  1. Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

    Science.gov (United States)

    Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphoro thioyloxy) benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-l...

  2. Examining consumer response to commodity-specific and broad-based promotion programs for fruits and vegetables using experimental economics

    OpenAIRE

    Rickard, Bradley J.; Liaukonyte, Jura; Kaiser, Harry M.; Richards, Timothy J.

    2010-01-01

    Generic promotion and advertising activities have traditionally been used to promote individual agricultural commodities. However, there is renewed interest in implementing a mandatory ―broad-based‖ promotion program for all fruits and vegetables, and this idea is highly controversial among those in the horticultural industry. Here we use data from an experiment that introduces subjects to various promotional efforts for fruits and vegetables to estimate the direct and indirect effects of adv...

  3. Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

    OpenAIRE

    Choy, F Y; Davidson, R G

    1980-01-01

    A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase act...

  4. The RSC Chromatin Remodeling Complex Bears an Essential Fungal-Specific Protein Module With Broad Functional Roles

    OpenAIRE

    Wilson, Boris; Erdjument-Bromage, Hediye; Tempst, Paul; Bradley R Cairns

    2006-01-01

    RSC is an essential and abundant ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae. Here we show that the RSC components Rsc7/Npl6 and Rsc14/Ldb7 interact physically and/or functionally with Rsc3, Rsc30, and Htl1 to form a module important for a broad range of RSC functions. A strain lacking Rsc7 fails to properly assemble RSC, which confers sensitivity to temperature and to agents that cause DNA damage, microtubule depolymerization, or cell wall stress (likely via tran...

  5. Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members.

    Science.gov (United States)

    Rigoldi, Federica; Spero, Ludovica; Dalle Vedove, Andrea; Redaelli, Alberto; Parisini, Emilio; Gautieri, Alfonso

    2016-07-19

    Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels. PMID:27327839

  6. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B. (DuPont)

    2010-03-08

    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

  7. High specific surface gold electrode on polystyrene substrate: Characterization and application as DNA biosensor.

    Science.gov (United States)

    Yang, Zhiliu; Liu, Yichen; Lu, Wei; Yuan, Qingpan; Wang, Wei; Pu, Qiaosheng; Yao, Bo

    2016-05-15

    In the past decades, many efforts have been made to improve the sensitivity and specificity of electrochemical DNA biosensors. However, it is still strongly required to develop disposable and reliable DNA biosensors for wide and practical application. In this article, we reported superior electrochemical properties of an integrated plastic-gold electrode (PGE) fabricated in-house by chemical plating on polystyrene substrate. PGEs were found having extremely high capacity of DNA immobilization compared with gold electrodes fabricated by standard sputtering based photolithography. Unique nano-structured surface was observed on PGEs through morphology techniques, which would to some extend give an explanation to higher capacity of DNA immobilization on PGEs. A probable mechanism of carboxylic acid produced on polystyrene substrate after exposure to UV irradiation was proposed and discussed for the first time. This biosensor was applied to detection and manipulate of DNA hybridization. Detection limit of 7.2×10(-11) M and 1-500 nM of linearity range was obtained.

  8. Naturally occurring ERAP1 haplotypes encode functionally distinct alleles with fine substrate specificity.

    Science.gov (United States)

    Reeves, Emma; Edwards, Christopher J; Elliott, Tim; James, Edward

    2013-07-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides for MHC class I presentation, influencing the degree and specificity of CD8(+) T cell responses. Single-nucleotide polymorphisms within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that single-nucleotide polymorphisms occur as distinct haplotypes in the human population and that these haplotypes encode functionally distinct ERAP1 alleles. Using a wide range of substrates, we are able to demonstrate that for any given substrate distinct ERAP1 alleles can be "normal," "hypofunctional," or "hyperfunctional" and that each allele has a trend bias toward one of these three activities. Thus, the repertoire of peptides presented at the cell surface for recognition by CTL is likely to depend on the precise combination of both MHC class I and ERAP1 alleles expressed within an individual, and has important implications for predisposition to disease. PMID:23733883

  9. Development of Specific Substrates for Hypocrea jecorina Cellulases

    DEFF Research Database (Denmark)

    Rasmussen, Tina Secher

    task since cellulose exists in different morphologies that can only be degraded efficiently by a battery of enzymes working together.3 One of the most efficient cellulosic systems is secreted by the fungus Hypocrea jecorina, which also has been employed in the industrial production of ethanol. However...... that they exhibit different substrate specificities. Therefore, a small library of derivatives of 2,4-dinitrophenyl cellobioside (2,4-DNPC) and 3,4-dinitrophenyl cellobioside (3,4-DNPC) was prepared. These derivatives contained a series of substituents (X and Y) located at the O4' and O6' position. Inspection...... chemistry or by amine coupling. The kinetic parameters of the library of compounds are to be determined....

  10. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  11. Programming of enzyme specificity by substrate mimetics: investigations on the Glu-specific V8 protease reveals a novel general principle of biocatalysis.

    Science.gov (United States)

    Wehofsky, N; Bordusa, F

    1999-01-25

    In this paper the universal validity of the substrate mimetic concept in enzymatic C-N ligations was expanded to anionic leaving groups based on the specificity determinants of Glu-specific endopeptidase from Staphylococcus aureus (V8 protease). In an empirical way a specific mimetic moiety was designed from simple structure-function relationship studies. The general function of the newly developed substrate mimetics to serve as an artificial recognition site for V8 protease have been examined by hydrolysis kinetic studies. Enzymatic peptide syntheses qualify the strategy of substrate mimetics as a powerful concept for programming the enzyme specificity in the direction of a more universal application of enzymes in the general area of biocatalysis. PMID:9989609

  12. Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Yip, P.; Cornell, K.; Riscoe, M.; Behr, J.; Guillerm, G.; Howell, P.

    2007-01-01

    The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  14. Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

    Science.gov (United States)

    Zhao, Fengchun; Tian, Yuan; Wang, Huimin; Liu, Jiye; Han, Xiao; Yang, Zhengyou

    2016-09-01

    Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides. PMID:27411546

  15. Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism.

    Science.gov (United States)

    Shen, Xing; Saburi, Wataru; Gai, Zuoqi; Kato, Koji; Ojima-Kato, Teruyo; Yu, Jian; Komoda, Keisuke; Kido, Yusuke; Matsui, Hirokazu; Mori, Haruhide; Yao, Min

    2015-06-01

    α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long β → α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure. PMID:26057678

  16. Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins

    OpenAIRE

    Sinha, Sharmistha; Lopes, Dahabada H. J.; Du, Zhenming; Pang, Eric S.; Shanmugam, Akila; Lomakin, Aleksey; Talbiersky, Peter; Tennstaedt, Annette; McDaniel, Kirsten; Bakshi, Reena; Kuo, Pei-Yi; Ehrmann, Michael; Benedek, George B.; Loo, Joseph A.; Klärner, Frank-Gerrit

    2011-01-01

    Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic a...

  17. Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

    OpenAIRE

    F. Bordusa

    2000-01-01

    This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The g...

  18. Structural and Functional Basis for Substrate Specificity and Catalysis of Levan Fructotransferase*

    Science.gov (United States)

    Park, Jinseo; Kim, Myung-Il; Park, Young-Don; Shin, Inchul; Cha, Jaeho; Kim, Chul Ho; Rhee, Sangkee

    2012-01-01

    Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the −1 and −2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements. PMID:22810228

  19. Structural and functional basis for substrate specificity and catalysis of levan fructotransferase.

    Science.gov (United States)

    Park, Jinseo; Kim, Myung-Il; Park, Young-Don; Shin, Inchul; Cha, Jaeho; Kim, Chul Ho; Rhee, Sangkee

    2012-09-01

    Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements. PMID:22810228

  20. Substrate-specific gene expression in Batrachochytrium dendrobatidis, the chytrid pathogen of amphibians.

    Directory of Open Access Journals (Sweden)

    Erica Bree Rosenblum

    Full Text Available Determining the mechanisms of host-pathogen interaction is critical for understanding and mitigating infectious disease. Mechanisms of fungal pathogenicity are of particular interest given the recent outbreaks of fungal diseases in wildlife populations. Our study focuses on Batrachochytrium dendrobatidis (Bd, the chytrid pathogen responsible for amphibian declines around the world. Previous studies have hypothesized a role for several specific families of secreted proteases as pathogenicity factors in Bd, but the expression of these genes has only been evaluated in laboratory growth conditions. Here we conduct a genome-wide study of Bd gene expression under two different nutrient conditions. We compare Bd gene expression profiles in standard laboratory growth media and in pulverized host tissue (i.e., frog skin. A large proportion of genes in the Bd genome show increased expression when grown in host tissue, indicating the importance of studying pathogens on host substrate. A number of gene classes show particularly high levels of expression in host tissue, including three families of secreted proteases (metallo-, serine- and aspartyl-proteases, adhesion genes, lipase-3 encoding genes, and a group of phylogenetically unusual crinkler-like effectors. We discuss the roles of these different genes as putative pathogenicity factors and discuss what they can teach us about Bd's metabolic targets, host invasion, and pathogenesis.

  1. Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.

    Directory of Open Access Journals (Sweden)

    Francesca Necchi

    Full Text Available Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS, is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b, whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA, whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2 is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40 able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein. Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

  2. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis

    Science.gov (United States)

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimIMtb. Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimIMtb is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimIMtb) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimIMtb is proposed. PMID:27353550

  3. Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Xiao; Chen, Ming; Zhang, Sheng; Yu, Zhi-Hong; Sun, Jin-Peng; Wang, Lina; Liu, Sijiu; Imasaki, Tsuyoshi; Takagi, Yuichiro; Zhang, Zhong-Yin (Indiana-Med)

    2012-02-08

    A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an 'inverse alanine scanning' combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.

  4. Comparing the glucuronidation capacity of the feline liver with substrate-specific glucuronidation in dogs

    NARCIS (Netherlands)

    van Beusekom, C D; Fink-Gremmels, J; Schrickx, J A

    2014-01-01

    This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis-Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compar

  5. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.

    Directory of Open Access Journals (Sweden)

    Hyung Jin Cha

    Full Text Available YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  6. Substrate specifity and inhibitors of polyphenol oxidase in aspect of darkening of fresh and frozen mushrooms (Agaricus bisporus (Lange Sing.

    Directory of Open Access Journals (Sweden)

    Janusz Czapski

    2013-12-01

    Full Text Available Activity of mushroom polyphenol oxidase (PPO toward 6 substrates and inhibitory effect of cysteine, 2-mercaptoethanol, benzoic acid and sodium metabisulphite were determined. The o-diphenols which appeared to be the best substrates were: catechin, DOPA (L-3,4-dihydro-xyphenylalanine and chlorogcnic acid. Affinity of PPO crude preparation substrates to enzyme, expressed as inverse value of Michaelis constant was lower then affinity of catechol. Inhibitory effect depended on specifity of inhibitors and their concentration. Electrophoretic patterns of PPO of mushrooms reveals slow and fast moving 4 isoforms when DOPA was used as a substrate, 2 bands for catechin and chlorogenic acid while only one band showed activity toward tyrosine and p-cresol.

  7. [Use of kenaf fibre in the elaboration of specific substrates for Pleurotus ostreatus (Jacq. ex Fr.) Kummer cultivation].

    Science.gov (United States)

    Pardo Giménez, Arturo; Perona Zamora, Ma Aquilina; Pardo Núñez, José

    2008-03-01

    In this study, the viability of the kenaf fibre use, alone or combined with cereal straw, vine shoots and olive mill dried waste, in the elaboration of specific substrates for the cultivation of Pleurotus ostreatus (Jacq. ex Fr.) Kummer, second mushroom in importance cultivated in Spain, is described. Furthermore, three different methods of preparation of the substrate have been considered in order to obtain selectivity for the growth and later fruiting of Pleurotus sporophore. As for the production parameters, the best results have been provided by the substrates that combined kenaf with straw and with vine shoots, being unfavourable the substrates based in just kenaf or combined with olive mill dried waste. As for the treatment applied to the materials, the immersion in water alone and subsequent pasteurization and thermophilic conditioning, together with the semi-anaerobic fermentation, has been favoured in front of the immersion in water with fungicide and later pasteurization.

  8. Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.

    Science.gov (United States)

    Sylwester, Andrew W; Mitchell, Bridget L; Edgar, John B; Taormina, Cara; Pelte, Christian; Ruchti, Franziska; Sleath, Paul R; Grabstein, Kenneth H; Hosken, Nancy A; Kern, Florian; Nelson, Jay A; Picker, Louis J

    2005-09-01

    Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4(+) and/or CD8(+) T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average approximately 10% of both the CD4(+) and CD8(+) memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8(+) T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans. PMID:16147978

  9. Structure based protein engineering of Bacillus stearothermophilus α-amylase: toward a new substrate specificity

    International Nuclear Information System (INIS)

    licheniformis crystal structure as initial model) it seems that Bacillus stearothermophilus α-amylase binding site is more complex with and insertion of 40 residues. Therefore the three dimensional structure is crucial to understand the specificity of the substrate of this enzyme which will be used to drive the design of mutation to introduce new properties for industrial purpose. (author)

  10. CD8+ T lymphocytes of patients with AIDS maintain normal broad cytolytic function despite the loss of human immunodeficiency virus-specific cytotoxicity

    International Nuclear Information System (INIS)

    In this study, the authors have investigated the potential mechanisms responsible for the loss of human immunodeficiency virus type 1 (HIV-1)-specific cytolytic activity in the advanced stages of HIV-1 infection. They have demonstrated that HIV-1-specific cytotoxic T lymphocytes are predominantly contained within the CD8+DR+ subset. Furthermore, they have shown by a redirected killing assay that there is a dichotomy between HIV-1-specific cytolytic activity and broad cytolytic potential since the cytolytic machinery of CD8+DR+ cells is still functioning even in patients with AIDS who have lost their HIV-1-specific cytolytic activity. In addition, by comparative analysis of these two types of cytolytic activity over time they have demonstrated a progressive loss of HIV-1-specific cytolytic activity in the advanced stages of the disease, whereas the cytolytic potential remained unchanged regardless of the clinical stage. On the basis of these results, they propose that the loss of HIV-1-specific cytolytic activity in HIV-1-infected individuals may result at least in part from a progressive decrease in the pool of HIV-1-specific cytotoxic T lymphocytes belonging to the CD8+DR+ subset whose ability to expand has been impaired

  11. Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium D-cysteine desulfhydrase.

    Directory of Open Access Journals (Sweden)

    Sakshibeedu R Bharath

    Full Text Available Salmonella typhimurium DCyD (StDCyD is a fold type II pyridoxal 5' phosphate (PLP-dependent enzyme that catalyzes the degradation of D-Cys to H(2S and pyruvate. It also efficiently degrades β-chloro-D-alanine (βCDA. D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC. Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS and L-cycloserine (LCS at resolutions ranging from 1.7 to 2.6 Å. The polypeptide fold of StDCyD consisting of a small domain (residues 48-161 and a large domain (residues 1-47 and 162-328 resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

  12. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G

    Science.gov (United States)

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.

    2016-01-01

    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  13. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

    Science.gov (United States)

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

    2011-02-01

    A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry.

  14. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    Science.gov (United States)

    Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. PMID:25111069

  15. Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments

    OpenAIRE

    Crits-Christoph, Alexander; Robinson, Courtney K.; Ma, Bing; Ravel, Jacques; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; Souza-Egipsy, Virginia; Casero, M. Cristina; DiRuggiero, Jocelyne

    2016-01-01

    Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and...

  16. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    Science.gov (United States)

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-03-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

  17. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541.

    Science.gov (United States)

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl

    2010-09-14

    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  18. Shotgun Isotope Array for Rapid, Substrate-Specific Detection of Microorganisms in a Microbial Community ▿ †

    OpenAIRE

    Tobino, Tomohiro; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

    2011-01-01

    The shotgun isotope array method has been proposed to be an effective new tool for use in substrate-specific microbe exploration without any prior knowledge of the community composition. Proof of concept was demonstrated by detection of acetate-degrading microorganisms in activated sludge and further verified by independent stable isotope probing (SIP).

  19. Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

    Science.gov (United States)

    Zheng, Linda; Tang, Joe; Clover, Gerard R G; Spackman, Merrin E; Freeman, Angela J; Rodoni, Brendan C

    2015-03-01

    A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.

  20. Alteration of substrate specificities of thermophilic α/β hydrolases through domain swapping and domain interface optimization

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Zhou; Honglei Wang; Yuhang Zhang; Le Gao; Yan Feng

    2012-01-01

    Protein domain swapping is an efficient way in protein functional evolution in vivo and also has been proved to be an effective strategy to modify the function of the multidomain proteins in vitro.To explore the potentials of domain swapping for alteration of the enzyme substrate specificities and the structure-function relationship of the homologous proteins,here we constructed two chimeras from a pair of thermophilic members of the α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain:a carboxylesterase from Archaeoglobus fulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyl chain lengths.We took two approaches to reduce the crossover disruptions when creating the chimeras:chose the residue which involved in the least contacts as the splicing site and optimized the newly formed domain interfaces of the chimeras by sitedirected mutations.Characterizations of AAM7 and PAR showed that these chimeras inherited the thermophilic property of both parents.In the aspect of substrate specificity,AAM7 and PAR showed highest activity towards short chain length substrate pNPC4 and middle chain length substrate pNPC8,similar to parent AFEST and apAPH,respectively.These results suggested that the substrate-binding domain is the dominant factor on enzyme substrate specificity,and the optimization of the newly formed domain interface is an important guarantee for successful domain swapping of proteins with low-sequence homology.

  1. Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase

    Energy Technology Data Exchange (ETDEWEB)

    Han,Q.; Gao, Y.; Robinson, H.; Li, J.

    2008-01-01

    Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant a-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate {beta}-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and {alpha}7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

  2. Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

    Directory of Open Access Journals (Sweden)

    Kishore Sarma

    2014-01-01

    Full Text Available ADP-glucose pyrophosphorylase (AGPase is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide.

  3. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang;

    2005-01-01

    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3......'-modified nucleoside analogs like 3'-azidothymidine ( AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction...... of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3'-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3'-substituents like the azido group in AZT...

  4. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    mechanism, in which the metal ion must bind prior to the substrate. Ni2+ and Cu2+ were inhibitors of ManA, which probably bind to the active site, but result in an enzyme unable to facilitate substrate binding. Numerous glycosidases have a carbohydrate binding domain (CBM) appended to the catalytic domain...... been identified in various enzymes, often based on crystal structures, and only few have been characterized in terms of structure-function relationship. Together SBS1 and SBS2 of barley α-amylase isozyme 1 probably represent the two most extensively studied SBSs. SBS2, largely governed by Tyr380, has...

  5. Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Katayama, Takane; Abou Hachem, Maher;

    2014-01-01

    signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize...... enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates...... (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic...

  6. Short-term in-vitro expansion improves monitoring and allows affordable generation of virus-specific T-cells against several viruses for a broad clinical application.

    Directory of Open Access Journals (Sweden)

    René Geyeregger

    Full Text Available Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8(+ and CD4(+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

  7. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  8. Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin; Howard, Andrew; Suzuki, Tomohiko; Moult, John; Herzberg, Osnat (Kochi); (IIT); (UMBI)

    2010-03-12

    a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

  9. Investigation of 1D Siliver Nanoparticle Arrays for Use as Molecule Concentration-Specific SERS Substrates

    Directory of Open Access Journals (Sweden)

    Jun Gao

    2013-01-01

    Full Text Available One-dimensional arrays of silver nanoparticles with a particle size of 10.5 nm and interparticle spacing of 8.5 nm were fabricated by depositing nanoparticles in gas phase on block copolymer self-assembled templates. The substrate showed a surface-enhanced Raman scattering (SERS enhancement factor as high as 1×107 with good reproducibility and stability. The dependence of the average enhancement factor and the SERS intensity on the packing density of the analyte molecules were investigated. For a tiny amount of analytes in the range of 1×10-14 to 3×10-13 mol/mm2, the SERS signal showed a linear dependence on the molecule packing density on a logarithmic scale, with a slope of about 1.25. The substrates are promising for quantitative detection of trace level molecules.

  10. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity.

    Science.gov (United States)

    Altmann, F; Schweiszer, S; Weber, C

    1995-02-01

    The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.

  11. General and Specific Effects on Cattell-Horn-Carroll Broad Ability Composites: Analysis of the Woodcock-Johnson III Normative Update Cattell-Horn-Carroll Factor Clusters across Development

    Science.gov (United States)

    Floyd, Randy G.; McGrew, Kevin S.; Barry, Amberly; Rafael, Fawziya; Rogers, Joshua

    2009-01-01

    Many school psychologists focus their interpretation on composite scores from intelligence test batteries designed to measure the broad abilities from the Cattell-Horn-Carroll theory. The purpose of this study was to investigate the general factor loadings and specificity of the broad ability composite scores from one such intelligence test…

  12. Phosphorylation-Dependent PIH1D1 Interactions Define Substrate Specificity of the R2TP Cochaperone Complex

    Directory of Open Access Journals (Sweden)

    Zuzana Hořejší

    2014-04-01

    Full Text Available The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs, RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

  13. Evaluation of Factor Xa-Specific Chromogenic Substrate Assays and the Determination of Pharmacokinetics of Fondaparinux.

    Science.gov (United States)

    Yuri, Maiko; Tabe, Yoko; Tsuchiya, Koji; Sadatsuki, Ryo; Aoki, Jun; Horii, Takashi; Iba, Toshiaki; Ohsaka, Akimichi

    2016-07-01

    Fondaparinux (FPX), a synthesized factor Xa inhibitor, is one of the most popular anticoagulants for the prevention of postoperative venous thromboembolism (VTE). Although routine monitoring is not required, the bleeding adverse events cannot be neglected, and the measurement of anti-Xa activity is expected to be monitored. The primary purpose of this study is to evaluate the performances of 2 chromogenic assays for the detection of anti-Xa activity. Furthermore, the pharmacokinetics of FPX was examined using chromogenic assays. Anti-Xa activity was measured using 2 FPX-based chromogenic substrates (S2222 and STA-Liquid Anti-Xa). The reproducibility, detection limits, linearity, and correlations between the substrates were examined using normal plasma doped with low and high concentrations of FPX formulation. In addition, anti-Xa activity in 235 clinical samples from 164 cases treated was measured, and the pharmacokinetics of FPX was evaluated. Both of the tested substrates were capable of accurately measuring the anti-Xa activity of FPX, with a lower limit of 0.05 μg/mL and a coefficient of variation of less than 10%. The repeated administration of FPX induced a gradual but significant increase in the anti-Xa activity, which was negatively correlated with body weight and estimated glomerular filtration rate. No significant correlation between the anti-Xa activity and the occurrence of postoperative VTE or bleeding event was observed. Anti-Xa activity can be successfully determined using 2 chromogenic assays and automated biochemical analyzers. The clinical significance of anti-Xa activity monitoring should be examined in the future study. PMID:26177660

  14. Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates.

    Science.gov (United States)

    Lin, Gang; Tsu, Christopher; Dick, Lawrence; Zhou, Xi K; Nathan, Carl

    2008-12-01

    The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome. PMID:18829465

  15. Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters.

    Science.gov (United States)

    Silverstein, Nechama; Ewers, David; Forrest, Lucy R; Fahlke, Christoph; Kanner, Baruch I

    2015-11-27

    Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

  16. Chemical tools for unraveling the substrate specificity of the lysine deacylase enzymes

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Olsen, Christian Adam

    The lysine deacylase (KDAC) enzymes catalyze hydrolytic removal of acyl functionalities from theε-amino group of lysine residues ina variety of proteins including histones, and KDAC-mediated deacetylation of proteins has been established as a key epigeneticandmetabolic regulator. Recent studies...... have highlighted lysine acetylation as a general post-translational modification (PTM), andagrowing list of non-histone proteins has been identified as substrates for the KDACs, thereby extending their potential impactoncellular function. Furthermore, other acyl groups (e.g., crotonyl, malonyl...

  17. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme

    Science.gov (United States)

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P.-Y.; Wang, Steven S.-S.

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells. PMID:27096746

  18. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    Science.gov (United States)

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  19. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

    Science.gov (United States)

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P-Y; Wang, Steven S-S

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

  20. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi [Marquette Univ., Milwaukee, WI (United States); Maurice, Martin [Marquette Univ., Milwaukee, WI (United States)

    2013-01-02

    Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2. AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH fromGranulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr299 and Arg307, are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr299 and Arg307 were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr299 and Arg307 serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172. The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

  1. UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity.

    Science.gov (United States)

    Beerens, Koen; Soetaert, Wim; Desmet, Tom

    2015-09-23

    UDP-sugar 4-epimerase (GalE) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins and is one of enzymes in the Leloir pathway. They have been shown to be important virulence factors in a number of Gram-negative pathogens and to be involved in the biosynthesis of different polysaccharide structures. The metabolic disease type III galactosemia is caused by detrimental mutations in the human GalE. GalE and related enzymes display unusual enzymologic, chemical, and stereochemical properties; including irreversible binding of the cofactor NAD and uridine nucleotide-induced activation of this cofactor. These epimerases have been found active on UDP-hexoses, the N-acetylated and uronic acid forms thereof as well as UDP-pentoses. As they are involved in different pathways and functions, a deeper understanding of the enzymes, and their substrate promiscuity and/or selectivity, could lead to drug and vaccine design as well as antibiotic and probiotic development. This review summarizes the research performed on UDP-sugar 4-epimerases' structure, mechanism and substrate promiscuity. PMID:26162744

  2. Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.

  3. The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

    Science.gov (United States)

    Bijelic, Aleksandar; Pretzler, Matthias; Molitor, Christian; Zekiri, Florime; Rompel, Annette

    2015-12-01

    Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed. PMID:26473311

  4. Substrate specific stimulation of NEIL1 by WRN but not the other human RecQ helicases

    DEFF Research Database (Denmark)

    Popuri, Venkateswarlu; Croteau, Deborah L; Bohr, Vilhelm A

    2010-01-01

    NEIL1, the mammalian homolog of Escherichia coli endonuclease VIII, is a DNA glycosylase that repairs ring-fragmented purines, saturated pyrimidines and several oxidative lesions like 5-hydroxyuracil, 5-hydroxycytosine, etc. Previous studies from our laboratory have shown that Werner Syndrome...... protein (WRN), one of the five human RecQ helicases, stimulates NEIL1 DNA glycosylase activity on oxidative DNA lesions. The goal of this study was to extend this observation and analyze the interaction between NEIL1 and all five human RecQ helicases. The DNA substrate specificity of the interaction...... between WRN and NEIL1 was also analyzed. The results indicate that WRN is the only human RecQ helicase that stimulates NEIL1 DNA glycosylase activity, and that this stimulation requires a double-stranded DNA substrate....

  5. The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity.

    Science.gov (United States)

    Hansen, Thomas; Reichstein, Bianca; Schmid, Roland; Schönheit, Peter

    2002-11-01

    An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which

  6. Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity.

    Directory of Open Access Journals (Sweden)

    Ellene H Mashalidis

    Full Text Available Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx, an enzyme that oxidizes pyridoxine 5'-phosphate (PNP or pyridoxamine 5'-phosphate (PMP to form pyridoxal 5'-phosphate (PLP. We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat and K(M for this reaction were 0.01 s(-1 and 360 µM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.

  7. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

    Science.gov (United States)

    Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

    2015-03-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

  8. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex*

    Science.gov (United States)

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.

    2010-01-01

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  9. Development of a monoclonal antibody specific to envelope domain III with broad-spectrum detection of all four dengue virus serotypes.

    Science.gov (United States)

    Kim, Sae-Hae; Kim, Yu Na; Truong, Thang Thua; Thu Thuy, Nguyen Thi; Mai, Le Quynh; Jang, Yong-Suk

    2016-05-13

    Dengue virus (DENV) is a mosquito-borne pathogen that annually infects more than 390 million people in 100 different countries. Symptoms of the viral infection include a relatively weak dengue fever to severe dengue hemorrhagic fever/dengue shock syndrome, which are mortal infectious diseases. As of yet, there is no commercially available vaccine or therapeutic for DENV. Currently, passive immunotherapy using DENV-specific antibody (Ab) is a considered strategy to treat DENV infection. Here, we developed a monoclonal Ab (mAb), EDIIImAb-61, specific to the DENV domain III of the envelope glycoprotein (EDIII) with broad-spectrum detection ability to all four DENV serotypes (DENV-1∼4) to use as a therapeutic Ab. Although EDIII contains non-immunodominant epitopes compared to domains I and II, domain III plays a critical role in host receptor binding. EDIIImAb-61 exhibited cross-reactive binding affinity to all four DENV serotypes that had been isolated from infected humans. To further characterize EDIIImAb-61 and prepare genes for large-scale production using a heterologous expression system, the sequence of the complementarity determining regions was analyzed after cloning the full-length cDNA genes encoding the heavy and light chain of the mAb. Finally, we produced Ab from CHO-K1 cells transfected with the cloned EDIIImAb-61 heavy and light chain genes and confirmed the binding ability of the Ab. Collectively, we conclude that EDIIImAb-61 itself and the recombinant Ab produced using the cloned heavy and light chain gene of EDIIImAb-61 is a candidate for passive immunotherapy against DENV infection. PMID:27059141

  10. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    International Nuclear Information System (INIS)

    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents

  11. Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

    Science.gov (United States)

    Liu, Jin; Han, Danxiang; Yoon, Kangsup; Hu, Qiang; Li, Yantao

    2016-04-01

    Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts. PMID:26919811

  12. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huiqing; Li, Xiaojing [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhao, Yuancong, E-mail: zhaoyc7320@163.com [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Li, Jingan; Chen, Jiang [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Yang, Ping, E-mail: yangping8@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Maitz, Manfred F. [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Max Bergmann Center of Biomaterials Dresden, Leibniz of Polymer Research Dresden, 01069 Dresden (Germany); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2015-08-30

    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  13. Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells

    International Nuclear Information System (INIS)

    A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDP-glucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector λgt11. The 2.4-kilobase cDNA contained an open reading frame of 1,593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of ∼ 55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of ∼ 3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase

  14. Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear

    Directory of Open Access Journals (Sweden)

    Vianna D.M.L.

    2003-01-01

    Full Text Available The periaqueductal gray (PAG has been traditionally considered to be an exit relay for defensive responses. Functional mapping of its subdivisions has advanced our knowledge of this structure, but synthesis remains difficult mainly because results from lesion and stimulation studies have not correlated perfectly. After using a strategy that combined both techniques and a reevaluation of the available literature on PAG function and connections, we propose here that freezing could be mediated by different PAG subdivisions depending on the presence of immediate danger or exposure to related signaling cues. These subdivisions are separate functional entities with distinct descending and ascending connections that are likely to play a role in different defensive responses. The existence of ascending connections also suggests that the PAG is not simply a final common path for defensive responses. For example, the possibility that indirect ascending connections to the cingulate cortex could play a role in the expression of freezing evoked by activation of the neural substrate of fear in the dorsal PAG has been considered.

  15. Problems in the experimental determination of substrate-specific H+/O ratios during respiration.

    Science.gov (United States)

    Hendler, R W; Shrager, R I

    1987-10-01

    Krab et al. (1984) have recently tried to resolve the long-standing controversy as to whether the mechanistic H+/O coupling ratio for electrons passing through sites II and III of the mammalian electron transport chain to O2 is 6 or 8. Using a mathematical model they concluded that the higher number reported by Costa et al. (1984) was an overestimate because of the unaccounted for delayed response of the O2 electrode. Responding to criticisms of Lehninger et al. (1985), they have recently used (Krab and Wikström, 1986) an improved mathematical model which shows that the higher number found by Costa et al. was probably due to an inadequate accounting for the effects of the proton leak process which accompanies the translocation process. The impression is left that the situation is now resolved in favor of the lower number. We agree that the procedures of Costa et al. do not properly account for the leak process, and provide further evidence in this paper of the magnitude of the problem. However, we disagree that the number 6.0, favored by Wikström et al., rests on any more solid experimental support. We provide evidence here for this conclusion and raise the question as to whether or not any unique, fixed, integral number exists for the H+/O ratio accompanying the oxidation of a particular substrate. PMID:2826412

  16. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    Science.gov (United States)

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. PMID:24355405

  17. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  18. Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperone

    International Nuclear Information System (INIS)

    FliT is a cytoplasmic flagellar type III substrate-specific export chaperone; it has been expressed, purified and crystallized and the crystals have been characterized by X-ray diffraction. The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2 Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination

  19. Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper.

    Science.gov (United States)

    Tegge, W; Frank, R; Hofmann, F; Dostmann, W R

    1995-08-22

    An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7654713

  20. Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

    Science.gov (United States)

    Toyota, Cory G; Berthold, Catrine L; Gruez, Arnaud; Jónsson, Stefán; Lindqvist, Ylva; Cambillau, Christian; Richards, Nigel G J

    2008-04-01

    The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions. PMID:18245280

  1. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases.

    Directory of Open Access Journals (Sweden)

    Eleni Ntokou

    Full Text Available Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering. RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence similarity and common target.

  2. Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli.

    OpenAIRE

    Heller, K B; Lin, E C; Wilson, T H

    1980-01-01

    The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly...

  3. Naturally occurring ERAP1 haplotypes encode functionally distinct alleles with fine substrate specificity

    OpenAIRE

    Reeves, Emma; Edwards, Christopher J.; Elliott, Tim; James, Edward

    2013-01-01

    The aminopeptidase, ERAP1, trims peptides for MHC class I presentation, influencing the degree and specificity of CD8+ T cell responses. Single nucleotide polymorphisms (SNP) within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that SNP occur as distinct haplotypes in the human population, and that these haplotypes e...

  4. Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities

    OpenAIRE

    Fesko, Kateryna

    2016-01-01

    Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable β-hydroxy-α-amino acids with complete stereocontrol at the α-carbon and moderate specificity at the β-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies ha...

  5. Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Petersen, G.E.; Sandrini, Michael;

    2003-01-01

    In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities......, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine...

  6. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

    Science.gov (United States)

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent

  7. Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

    Science.gov (United States)

    Harris, M E; Kazantsev, A V; Chen, J L; Pace, N R

    1997-06-01

    Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme. PMID:9174092

  8. Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.

    Science.gov (United States)

    Arndt, Joseph W; Chai, Qing; Christian, Todd; Stevens, Raymond C

    2006-03-14

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

  9. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    Science.gov (United States)

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

  10. Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

    Science.gov (United States)

    Libantová, Jana; Kämäräinen, Terttu; Moravcíková, Jana; Matusíková, Ildikó; Salaj, Jan

    2009-05-01

    The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated. PMID:18437530

  11. Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures.

    Science.gov (United States)

    Devlin, Shauna; Meneely, Julie P; Greer, Brett; Campbell, Katrina; Vasconcelos, Vitor; Elliott, Christopher T

    2014-05-01

    A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989. PMID:24720955

  12. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob;

    2015-01-01

    Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence......-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore...... to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence...

  13. Excimer laser texturing of natural composite polymer surfaces for studying cell-to-substrate specific response

    Science.gov (United States)

    Dinca, V.; Alloncle, P.; Delaporte, P.; Ion, V.; Rusen, L.; Filipescu, M.; Mustaciosu, C.; Luculescu, C.; Dinescu, M.

    2015-10-01

    Surface modifications of biocompatible materials are among the main factors used for enhancing and promoting specific cellular activities (e.g. spreading, adhesion, migration, and differentiation) for various types of medical applications such as implants, microfluidic devices, or tissue engineering scaffolds. In this work an excimer laser at 193 nm was used to fabricate chitosan-collagen roughness gradients. The roughness gradients were obtained in one step by applying single laser pulses and sample tilting. Fourier transform infrared spectroscopy measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and spectro-ellipsometry (SE) were used for sample characterization. The goal is to determine the optimal morpho-chemical characteristics of these structures for in vitro tailoring of protein adsorption and cell behavior. The response induced by the roughness gradient onto various cell lines (i.e. L 929 fibroblasts, HEP G2 hepatocytes, OLN 93 oligodendrocytes, M63 osteoblasts) and bovine serum albumin (BSA) protein absorption was investigated.

  14. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III

    Science.gov (United States)

    Richard-Fogal, Cynthia L.; Francisco, Brian San; Frawley, Elaine R.; Kranz, Robert G.

    2011-01-01

    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E.coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E.coli periplasmic DsbC/DsbD thiol-reduction pathway. PMID:21945855

  15. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    NARCIS (Netherlands)

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(meth

  16. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    Energy Technology Data Exchange (ETDEWEB)

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  17. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  18. Specific Neuron Placement on Gold and Silicon Nitride-Patterned Substrates through a Two-Step Functionalization Method.

    Science.gov (United States)

    Mescola, Andrea; Canale, Claudio; Prato, Mirko; Diaspro, Alberto; Berdondini, Luca; Maccione, Alessandro; Dante, Silvia

    2016-06-28

    considered a good starting point to develop alternatives to the traditional chip coatings to provide selective and specific neuron-substrate adhesion. PMID:27268249

  19. Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

  20. A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

    Science.gov (United States)

    Mason, Deborah L; Mallampalli, Monica P; Huyer, Gregory; Michaelis, Susan

    2003-06-01

    Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6(ins)), which appears to be necessary and sufficient for proteolytic cleavage, since L6(ins) can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p. Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity. Instead, the L6(ins) appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance. Although some of these L6(ins) mutations block processing, there is no correlation between processing and substrate specificity. The activity profiles of the Ycf1p L6(ins) mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity. A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame. PMID:12796304

  1. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    Directory of Open Access Journals (Sweden)

    Silvija Miosic

    Full Text Available During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin and two (cyanidin hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring, whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  2. Substrate Specificity and Ionic Regulation of GlnPQ from Lactococcus lactis. An ATP-Binding Cassette Transporter with Four Extracytoplasmic Substrate-Binding Domains

    NARCIS (Netherlands)

    Schuurman-Wolters, Gea K.; Poolman, Bert

    2005-01-01

    We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmi

  3. Broad-based nutritional supplementation in 3xTg mice corrects mitochondrial function and indicates sex-specificity in response to Alzheimer's disease intervention.

    Science.gov (United States)

    Wolf, Andrew B; Braden, B Blair; Bimonte-Nelson, Heather; Kusne, Yael; Young, Nicole; Engler-Chiurazzi, Elizabeth; Garcia, Alexandra N; Walker, Douglas G; Moses, Guna S D; Tran, Hung; LaFerla, Frank; Lue, LihFen; Emerson Lombardo, Nancy; Valla, Jon

    2012-01-01

    Nutrition has been highlighted as a potential factor in Alzheimer's disease (AD) risk and decline and has been investigated as a therapeutic target. Broad-based combination diet therapies have the potential to simultaneously effect numerous protective and corrective processes, both directly (e.g., neuroprotection) and indirectly (e.g., improved vascular health). Here we administered either normal mouse chow with a broad-based nutritional supplement or mouse chow alone to aged male and female 3xTg mice and wildtype (WT) controls. After approximately 4 months of feeding, mice were given a battery of cognitive tasks and then injected with a radiolabeled glucose analog. Brains were assessed for differences in regional glucose uptake and mitochondrial cytochrome oxidase activity, AD pathology, and inflammatory markers. Supplementation induced behavioral changes in the 3xTg, but not WT, mice, and the mode of these changes was influenced by sex. Subsequent analyses indicated that differential response to supplementation by male and female 3xTg mice highlighted brain regional strategies for the preservation of function. Several regions involved have been shown to mediate responses to steroid hormones, indicating a mechanism for sex-based vulnerability. Thus, these findings may have broad implications for the human response to future therapeutics.

  4. Determination of residues responsible for substrate and product specificity of Solanum habrochaites short-chain cis-prenyltransferases.

    Science.gov (United States)

    Kang, Jin-Ho; Gonzales-Vigil, Eliana; Matsuba, Yuki; Pichersky, Eran; Barry, Cornelius S

    2014-01-01

    Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species. PMID:24254315

  5. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    Science.gov (United States)

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  6. Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons.

    Science.gov (United States)

    Matsuda, Miho; Rand, Kinneret; Palardy, Greg; Shimizu, Nobuyuki; Ikeda, Hiromi; Dalle Nogare, Damian; Itoh, Motoyuki; Chitnis, Ajay B

    2016-09-01

    We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

  7. Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons.

    Science.gov (United States)

    Matsuda, Miho; Rand, Kinneret; Palardy, Greg; Shimizu, Nobuyuki; Ikeda, Hiromi; Dalle Nogare, Damian; Itoh, Motoyuki; Chitnis, Ajay B

    2016-09-01

    We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons. PMID:27510968

  8. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

    Science.gov (United States)

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

    2014-07-01

    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. PMID:24687155

  9. The crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity.

    Science.gov (United States)

    Yaoi, Katsuro; Kondo, Hidemasa; Hiyoshi, Ayako; Noro, Natsuko; Sugimoto, Hiroshi; Tsuda, Sakae; Miyazaki, Kentaro

    2009-09-01

    Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-beta-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode. Here, we determined the crystal structure of XEG at 2.5 A resolution, and compared it to a previously determined structure of OXG-RCBH. For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes. However, there are notable differences at subsite positions -1 and +2. OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate. At the -1 site in OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the -1 site is occupied by Tyr457 in XEG. To confirm the contribution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG. The wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites. Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG. PMID:19682300

  10. Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

    Science.gov (United States)

    Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

    2015-04-15

    In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico. PMID:25797391

  11. Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Keun; Murugesan, Senthilkumar [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-06-15

    Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg{sup -1} protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg{sup -1} protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg{sup -1} protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg{sup -1} protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg{sup -1} protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions.

  12. DCs Pulsed with Novel HLA-A2-Restricted CTL Epitopes against Hepatitis C Virus Induced a Broadly Reactive Anti-HCV-Specific T Lymphocyte Response

    OpenAIRE

    Zhongsheng Guo; Henghui Zhang; Huiying Rao; Dong Jiang; Xu Cong; Bo Feng(Zhejiang Institute of Modern Physics, Zhejiang University, 38 Zheda Road Hangzhou, 310027 P.R. China); Jianghua Wang; Lai Wei; Hongsong Chen

    2012-01-01

    OBJECTIVE: To determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripher...

  13. Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism.

    Science.gov (United States)

    Fischer, Thilo C; Halbwirth, Heidrun; Meisel, Barbara; Stich, Karl; Forkmann, Gert

    2003-04-15

    Treatment with the dioxygenase inhibitor prohexadione-Ca leads to major changes in the flavonoid metabolism of apple (Malus domestica) and pear (Pyrus communis) leaves. Accumulation of unusual 3-deoxyflavonoids is observed, which have been linked to an enhanced resistance toward fire blight. The committed step in this pathway is the reduction of flavanones. Crude extracts from leaves are able to perform this reaction. There was previous evidence that DFR enzymes of certain plants possess additional flavanone 4-reductase (FNR) activity. Such an FNR activity of DFR enzymes is proved here by heterologous expression of the enzymes. The heterologously expressed DFR/FNR enzymes of Malus and Pyrus possess distinct differences in substrate specificities despite only minor differences of the amino acid sequences. Kinetic studies showed that dihydroflavonols generally are the preferred substrates. However, with the observed substrate specificities the occurrence of 3-deoxyflavonoids in vivo after application of prohexadione-Ca can be explained.

  14. Substrate specificity of the OqxAB multidrug resistance pump in Escherichia coli and selected enteric bacteria

    DEFF Research Database (Denmark)

    Hansen, Lars Hestbjerg; Jensen, Lars Bogø; Sørensen, Heidi Iskou;

    2007-01-01

    a variety of substrates in E. coli. These included animal growth promoters, antimicrobials, disinfectants and detergents. pOLA52 could readily be transferred to enterobacterial pathogens. Transconjugants showed reduced susceptibility towards chloramphenicol, ciprofloxacin and olaquindox. Conclusions...

  15. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations

    OpenAIRE

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A.; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-01

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were...

  16. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

    Science.gov (United States)

    Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

    2013-01-01

    Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

  17. New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Aduri, Nanda Gowtham; Jensen, Johanne Mørch;

    2014-01-01

    Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of E. coli POTs YdgR, YhiP and YjdL were investigated by means of a la...

  18. Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

    Science.gov (United States)

    Shirafuji, Hiroaki; Yazaki, Ryu; Shuto, Yozo; Yanase, Tohru; Kato, Tomoko; Ishikura, Youji; Sakaguchi, Zenjiro; Suzuki, Moemi; Yamakawa, Makoto

    2015-12-01

    TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

  19. First Membrane Proximal External Region-Specific Anti-HIV1 Broadly Neutralizing Monoclonal IgA1 Presenting Short CDRH3 and Low Somatic Mutations.

    Science.gov (United States)

    Benjelloun, Fahd; Oruc, Zeliha; Thielens, Nicole; Verrier, Bernard; Champier, Gael; Vincent, Nadine; Rochereau, Nicolas; Girard, Alexandre; Jospin, Fabienne; Chanut, Blandine; Genin, Christian; Cogné, Michel; Paul, Stephane

    2016-09-01

    Mucosal HIV-1-specific IgA have been described as being able to neutralize HIV-1 and to block viral transcytosis. In serum and saliva, the anti-HIV IgA response is predominantly raised against the envelope of HIV-1. In this work, we describe the in vivo generation of gp41-specific IgA1 in humanized α1KI mice to produce chimeric IgA1. Mice were immunized with a conformational immunogenic gp41-transfected cell line. Among 2300 clones screened by immunofluorescence microscopy, six different gp41-specific IgA with strong recognition of gp41 were identified. Two of them have strong neutralizing activity against primary HIV-1 tier 1, 2, and 3 strains and present a low rate of somatic mutations and autoreactivity, unlike what was described for classical gp41-specific IgG. Epitopes were identified and located in the hepted repeat 2/membrane proximal external region. These Abs could be of interest in prophylactic treatment to block HIV-1 penetration in mucosa or in chronically infected patients in combination with antiretroviral therapy to reduce viral load and reservoir. PMID:27481846

  20. Comparison of a Broad-Based Screen versus Disorder-Specific Screen in Detecting Young Children with an Autism Spectrum Disorder

    Science.gov (United States)

    Wiggins, Lisa D; Piazza, Vivian; Robins, Diana L

    2014-01-01

    The goals of our study were to (a) compare agreement between autism spectrum disorder diagnosis and outcome of the Modified Checklist for Autism in Toddlers and Parents Evaluation of Developmental Status in a sample of toddlers and (b) examine specific concerns noted for toddlers who screened negative on the Modified Checklist for Autism in…

  1. DCs pulsed with novel HLA-A2-restricted CTL epitopes against hepatitis C virus induced a broadly reactive anti-HCV-specific T lymphocyte response.

    Directory of Open Access Journals (Sweden)

    Zhongsheng Guo

    Full Text Available OBJECTIVE: To determine the capacity of dendritic cells (DCs loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance. Mature DCs, derived in vitro from CD14(+ monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry. RESULTS: We identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs. Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response. CONCLUSIONS: Seven novel and

  2. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice

    Science.gov (United States)

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

    2016-01-01

    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction. PMID:26305669

  3. Comparison of a Broad-Based Screen versus Disorder-Specific Screen in Detecting Young Children with an Autism Spectrum Disorder

    OpenAIRE

    Wiggins, Lisa D.; Piazza, Vivian; Robins, Diana L.

    2012-01-01

    The goals of our study were to (a) compare agreement between autism spectrum disorder diagnosis and outcome of the Modified Checklist for Autism in Toddlers and Parents Evaluation of Developmental Status in a sample of toddlers and (b) examine specific concerns noted for toddlers who screened negative on the Modified Checklist for Autism in Toddlers or Parents Evaluation of Developmental Status but were later diagnosed with autism spectrum disorder. Participants were administered the Modified...

  4. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations.

    Science.gov (United States)

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-20

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed.

  5. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations.

    Science.gov (United States)

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-01

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed. PMID:25603968

  6. A Coherent Study of Emission Lines from Broad-Band Photometry: Specific Star-Formation Rates and [OIII]/H{\\beta} Ratio at 3 < z < 6

    CERN Document Server

    Faisst, A L; Hsieh, B C; Laigle, C; Salvato, M; Tasca, L; Cassata, P; Davidzon, I; Ilbert, O; Fevre, O Le; Masters, D; McCracken, H J; Steinhardt, C; Silverman, J D; De Barros, S; Hasinger, G; Scoville, N Z

    2016-01-01

    We measure the H{\\alpha} and [OIII] emission line properties as well as specific star-formation rates (sSFR) of spectroscopically confirmed 33 cannot be fully explained in a picture of cold accretion driven growth. We find a progressively increasing [OIII]{\\lambda}5007/H{\\beta} ratio out to z~6, consistent with the ratios in local galaxies selected by increasing H{\\alpha} EW (i.e., sSFR). This demonstrates the potential of using "local high-z analogs" to investigate the spectroscopic properties and relations of galaxies in the re-ionization epoch.

  7. pKa modulation of the acid/base catalyst within GH32 and GH68: a role in substrate/inhibitor specificity?

    Directory of Open Access Journals (Sweden)

    Shuguang Yuan

    Full Text Available Glycoside hydrolases of families 32 (GH32 and 68 (GH68 belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates and fructosyltransferases (sucrose/fructans as donor and acceptor substrates. In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif rather than the previously proposed Tyr motif (not conserved provides the proton to increase the pKa of the acid-base catalyst.

  8. Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

    Institute of Scientific and Technical Information of China (English)

    WU Hai-hua; ZHU Kun-yan; GUO Ya-ping; ZHANG Xiao-min; MA En-bo

    2008-01-01

    Specific activity, substrate specificity, and kinetic parameters (Km and Vmax) of glutathione S-transferases (GSTs) towards three substrates, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzene chloride (pNBC) were investigated in six tissues (foregut, midgut, hindgut, fat body, hemolymph, and muscle) of Oxya chinensis. In addition, the inhibition in vitro (ethacrynic acid, and Cibacron Blue 3GA) of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body > midgut > hindgut > muscle > foregut > hemolymph both in females and males. Glutathione 5-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0-fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration (IC50) values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.

  9. Structural characterization of mouse neutrophil serine proteases and identification of their substrate specificities: relevance to mouse models of human inflammatory diseases.

    Science.gov (United States)

    Kalupov, Timofey; Brillard-Bourdet, Michèle; Dadé, Sébastien; Serrano, Hélène; Wartelle, Julien; Guyot, Nicolas; Juliano, Luiz; Moreau, Thierry; Belaaouaj, Azzaq; Gauthier, Francis

    2009-12-01

    It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases.

  10. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  11. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    OpenAIRE

    Fraaije, Marco W.; Veeger, Cees; van Berkel, Willem J. H.

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyph...

  12. Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

    Science.gov (United States)

    Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C; Kelleher, Neil L; Shen, Ben

    2006-10-01

    C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically. PMID:16887797

  13. Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, James; Murphy, Loretta M.; Conners, Rebecca; Sessions, Richard B.; Gamblin, Steven J. (Wales); (Bristol Med Sci); (NIMR)

    2010-09-21

    Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.

  14. Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

    Directory of Open Access Journals (Sweden)

    Mayer Kimberly M

    2007-01-01

    Full Text Available Abstract Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174 changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT or 74, 86, 141, 174 (4-MUT were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.

  15. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

    Science.gov (United States)

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2016-07-01

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

  16. The 2010 Broad Prize

    Science.gov (United States)

    Education Digest: Essential Readings Condensed for Quick Review, 2011

    2011-01-01

    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  17. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity*

    Science.gov (United States)

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H.; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R.; Rubin, Eric J.; Bachovchin, William; Goldberg, Alfred L.

    2015-01-01

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

  18. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    Science.gov (United States)

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  19. Subcloning of the enterobactin biosynthetic gene entB: Expression, purification, characterization, and substrate specificity of isochorismatase

    International Nuclear Information System (INIS)

    The Escherichia coli entB, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 μM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway

  20. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  1. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity.

    Science.gov (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha

    2011-01-15

    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  2. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applicatons in agriculture

    Energy Technology Data Exchange (ETDEWEB)

    Dirk, Lynnette M.A.; Schmidt, Jack J.; Cai, Yiying; Barnes, Jonathan C.; Hanger, Katherine M.; Nayak, Nihar R.; Williams, Mark A.; Grossman, Robert B.; Houtz, Robert L.; Rodgers, David W. (Kentucky)

    2008-07-28

    The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 {angstrom} (1 {angstrom}=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr{sup 178} as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr{sup 2} in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr{sup 178} to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr{sup 178} can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  3. Specificity of the SUV4-20H1 and SUV4-20H2 protein lysine methyltransferases and methylation of novel substrates.

    Science.gov (United States)

    Weirich, Sara; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-06-01

    The SUV4-20H1 and SUV4-20H2 enzymes methylate histone H4 at K20, and they have overlapping and distinct biological effects. Here, by in vitro methylation studies we confirmed that both the murine SUV4-20H enzymes strongly favor the monomethylated H4K20 peptide substrate. We also show that both enzymes only generate dimethylated H4K20 products. We determined the substrate sequence recognition motif of both enzymes using SPOT peptide arrays showing that SUV4-20H1 recognizes an (RY)-Kme1-(IVLM)-(LFI)-X-D sequence. In contrast, SUV4-20H2 shows less specificity and recognizes an X-Kme1-(IVLMK)-(LVFI)-X-(DEV) sequence, which is partially overlapping with SUV4-20H1 but has relaxed specificity at the -1 and +4 positions (if the target H4K20me1 is positon 0). Based on our data, we identify novel peptide substrates for SUV4-20H1 (K1423 of Zinc finger protein castor homolog 1) and SUV4-20H2 (K1423 of Zinc finger protein castor homolog 1, K215 of Protein Mis18-beta and K308 of Centromere protein U). All these lysine residues were already identified to be methylated in human cells, but the responsible PKMT was not known. In addition, we also tested the activity of SUV4-20H enzymes on ERK1, which was recently reported to be methylated by SUV4-20H1 at K302 and K361. However the sequences surrounding both methylation sites do not fit to the specificity profile of SUV4-20H1 and we could not detect methylation of ERK1 by any of the SUV4-20H enzymes. The possible reasons of this discrepancy and its consequences are discussed. PMID:27105552

  4. Extension of Drosophila lifespan by cinnamon through a sex-specific dependence on the insulin receptor substrate chico.

    Science.gov (United States)

    Schriner, Samuel E; Kuramada, Steven; Lopez, Terry E; Truong, Stephanie; Pham, Andrew; Jafari, Mahtab

    2014-12-01

    Cinnamon is a spice commonly used worldwide to flavor desserts, fruits, cereals, breads, and meats. Numerous health benefits have been attributed to its consumption, including the recent suggestion that it may decrease blood glucose levels in people with diabetes. Insulin signaling is an integral pathway regulating the lifespan of laboratory organisms, such as worms, flies, and mice. We posited that if cinnamon truly improved the clinical signs of diabetes in people that it would also act on insulin signaling in laboratory organisms and increase lifespan. We found that cinnamon did extend lifespan in the fruit fly, Drosophila melanogaster. However, it had no effect on the expression levels of the 3 aging-related Drosophila insulin-like peptides nor did it alter sugar, fat, or soluble protein levels, as would be predicted. In addition, cinnamon exhibited no protective effects in males against oxidative challenges. However, in females it did confer a protective effect against paraquat, but sensitized them to iron. Cinnamon provided no protective effect against desiccation and starvation in females, but sensitized males to both. Interestingly, cinnamon protected both sexes against cold, sensitized both to heat, and elevated HSP70 expression levels. We also found that cinnamon required the insulin receptor substrate to extend lifespan in males, but not females. We conclude that cinnamon does not extend lifespan by improving stress tolerance in general, though it does act, at least in part, through insulin signaling. PMID:25456850

  5. α--AMYLASES OF Aspergillus flavus var. oryzae AND Bacillus subtilis: THE SUBSTRATE SPECIFICITY AND RESISTANCE TO A NUMBER OF CHEMICALLY ACTIVE SUBSTANCES

    Directory of Open Access Journals (Sweden)

    K. V. Avdiyuk

    2013-06-01

    Full Text Available The ability of Aspergillus flavus var. oryzae 80428 and Bacillus subtilis 147 α-amylases to split different carbohydrate-containing substrates, such as maltose, sucrose, trehalose, dextrin, α- and β-cyclodextrin, amylose, amylopectin, glycogen, pullulan, soluble starch, insoluble starch, corn starch, wheat starch, dextran 500 has been studied. It was shown that investigated enzymes differ by substrate specificity. α-Amylase of A. flavus var. oryzae 80428 rapidly hydrolysed soluble potato and wheat starch, while the α-amylase of B. subtilis 147 — only wheat starch. Both enzymes don’t cleave maltose, α-cyclodextrin and dextran 500. A. flavus var. oryzae 80428 α-amylase display very small ability to hydrolyze pullulan, while α-amylase of B. subtilis 147 it does not act in general. The lowest values of Michaelis constant for both enzymes at splitting of glycogen have been obtained, indicating that enzymes have the greatest affinity to this substrate. The studies of influence of chemically active substances on activity of A. flavus var. oryzae 80428 and B. subtilis 147 ?-amylases show there are resistant to urea, deoxycholic acid, Tween-80, Triton X-100 and hydrogen peroxide. It’s indicate the enzymes tested may be competitive in compare with earlier described in literature enzymes. The obtained results give a possibility to propose in future usage these enzymes in different fields of industry, foremost in detergent industry.

  6. Combining substrate specificity analysis with support vector classifiers reveals feruloyl esterase as a phylogenetically informative protein group

    DEFF Research Database (Denmark)

    Olivares Hernandez, Roberto; Sunner, Hampus; Frisvad, Jens Christian;

    2010-01-01

    Background Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able...

  7. FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response

    Science.gov (United States)

    Daskalova, A.; Nathala, Chandra S. R.; Kavatzikidou, P.; Ranella, A.; Szoszkiewicz, R.; Husinsky, W.; Fotakis, C.

    2016-09-01

    The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell's responses to substrate morphology. Mice fibroblasts migration was monitored after 3 days cultivation period using FESEM. We found that fibroblasts cells tend to migrate and adhere along the laser modified zones. The performed study proved that the immobilized collagen based biofilms suite as a template for successful fibroblasts cell guidance and orientation. Fs laser induced morphological modification of biomimetic materials exhibit direct control over fibroblasts behaviour due to induced change in their wettability state.

  8. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

    Science.gov (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-08-19

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  9. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    Science.gov (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  10. Crystal Structure of the Homo sapiens Kynureninase-3-Hydroxyhippuric Acid Inhibitor Complex: Insights into the Molecular Basis Of Kynureninase Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Lima,Santiago; Kumar,Sunil; Gawandi,Vijay; Momany,Cory; Phillips,Robert S.; (Georgia)

    2009-02-23

    Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD{sup +}. This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease. We have synthesized a novel kynureninase inhibitor, 3-hydroxyhippurate, cocrystallized it with human kynureninase, and solved the atomic structure. On the basis of an analysis of the complex, we designed a series of His-102, Ser-332, and Asn-333 mutants. The H102W/N333T and H102W/S332G/N333T mutants showed complete reversal of substrate specificity between 3-hydroxykynurenine and L-kynurenine, thus defining the primary residues contributing to substrate specificity in kynureninases.

  11. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

    Science.gov (United States)

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-06-01

    Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases. PMID:17557809

  12. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

    Directory of Open Access Journals (Sweden)

    Hong Wa Yung

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  13. Protein-carbohydrate interactions defining substrate specificity in Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases as dissected by mutational analysis.

    Science.gov (United States)

    Piotukh, K; Serra, V; Borriss, R; Planas, A

    1999-12-01

    The carbohydrate-binding site of Bacillus macerans 1,3-1, 4-beta-D-glucan 4-glucanohydrolase has been analyzed through a mutational analysis to probe the role of protein-carbohydrate interactions defining substrate specificity. Amino acid residues involved in substrate binding were proposed on the basis of a modeled enzyme-substrate complex [Hahn, M., Keitel, T., and Heinemann, U. (1995) Eur. J. Biochem. 232, 849-859]. The effects of the mutations at 15 selected residues on catalysis and binding were determined by steady-state kinetics using a series of chromogenic substrates of different degree of polymerization to assign the individual H-bond and hydrophobic contributions to individual subsites in the binding site cleft. The glucopyranose rings at subsites -III and -II are tightly bound by a number of H-bond interactions to Glu61, Asn24, Tyr92, and Asn180. From k(cat)/K(M) values, single H-bonds account for 1.8-2.2 kcal mol(-)(1) transition-state (TS) stabilization, and a charged H-bond contributes up to 3.5 kcal mol(-)(1). Glu61 forms a bidentated H-bond in subsites -III and -II, and provides up to 6.5 kcal mol(-)(1) TS stabilization. With a disaccharide substrate that fills subsites -I and -II, activation kinetics were observed for the wild-type and mutant enzymes except for mutations on Glu61, pointing to an important role of the bidentate interaction of Glu61 in two subsites. Whereas removal of the hydroxyl group of Tyr121, initially proposed to hydrogen-bond with the 2OH of Glcp-I, has essentially no effect (Y121F mutant), side-chain removal (Y121A mutant) gave a 100-fold reduction in k(cat)/K(M) and a 10-fold lower K(I) value with a competitive inhibitor. In subsite -IV, only a stacking interaction with Tyr22 (0.7 kcal mol(-)(1) TS stabilization) is observed. PMID:10587432

  14. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

    DEFF Research Database (Denmark)

    Auxilien, Sylvie; El Khadali, Fatima; Rasmussen, Anette;

    2007-01-01

    Members of the archease superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing. Archease have therefore been predicted to play a modulator or chaperone role in selected steps...... of DNA or RNA metabolism, although the roles of archeases remain to be established experimentally. Here we report the function of one of these archeases from the hyperthermophile Pyrococcus abyssi. The corresponding gene (PAB1946) is located in a bicistronic operon immediately upstream from a second open...... reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease...

  15. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David, E-mail: dave@scripps.edu [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2011-06-01

    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC{sub 50} values in the nanomolar range.

  16. Structural studies of ROK fructokinase YdhR from Bacillus subtilis : insights into substrate binding and fructose specificity.

    Energy Technology Data Exchange (ETDEWEB)

    Nocek, B.; Stein, A.; Jedrzejczak, R.; Cuff, M.; Li, H.; Volkart, L.; Joachimiak, A.; Biosciences Division

    2011-02-18

    The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the 'ROK' (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar {alpha}/{beta} domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

  17. Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide.

    Science.gov (United States)

    Hamm, Michelle L; McFadden, Emily J; Ghio, Michael; Lindell, Maria A M; Gerien, Kenneth S; O'Handley, Suzanne F

    2016-04-15

    The bacterial repair enzyme MutT hydrolyzes the damaged nucleotide OdGTP (the 5'-triphosphate derivative of 8-oxo-2'-deoxyguanosine; OdG), which is a known mutagen and has been linked to antibacterial action. Previous work has indicated important roles for the C8-oxygen, N7-hydrogen, and C2-exocyclic amine during OdGTP recognition by MutT. In order to gain a more nuanced understanding of the contribution of these three sites to the overall activity of MutT, we determined the reaction parameters for dGTP, OdGTP, and nine of their analogues using steady state kinetics. Our results indicate that overall high reaction efficiencies can be achieved despite altering any one of these sites. However, altering two or more sites leads to a significant decrease in efficiency. The data also suggest that, similar to another bacterial OdG repair enzyme, MutM, a specific carbonyl in the enzyme can not only promote activity by forming an active site hydrogen bond with the N7-hydrogen of OdGTP, but can also hinder activity through electrostatic repulsion with the N7-lone pair of dGTP.

  18. The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

    Science.gov (United States)

    Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

    2016-07-19

    It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell. PMID:27225936

  19. High Resolution Structures of Periplasmic Glucose-binding Protein of Pseudomonas putida CSV86 Reveal Structural Basis of Its Substrate Specificity.

    Science.gov (United States)

    Pandey, Suman; Modak, Arnab; Phale, Prashant S; Bhaumik, Prasenjit

    2016-04-01

    Periplasmic substrate-binding proteins (SBPs) bind to the specific ligand with high affinity and mediate their transport into the cytoplasm via the cognate inner membrane ATP-binding cassette proteins. Because of low sequence identities, understanding the structural basis of substrate recognition by SBPs has remained very challenging. There are several structures available for the ligand-bound sugar SBPs, but very few unliganded structures are reported. No structural data are available for sugar SBPs fromPseudomonassp. to date. This study reports the first high resolution crystal structures of periplasmic glucose-binding protein fromPseudomonas putidaCSV86 (ppGBP) in unliganded form (2.5 Å) and complexed with glucose (1.25 Å) and galactose (1.8 Å). Asymmetric domain closure of ppGBP was observed upon substrate binding. The ppGBP was found to have an affinity of ∼ 0.3 μmfor glucose. The structural analysis showed that the sugars are bound to the protein mainly by hydrogen bonds, and the loss of two strong hydrogen bonds between ppGBP and galactose compared with glucose may be responsible for lowering its affinity toward galactose. The higher stability of ppGBP-glucose complex was also indicated by an 8 °C increase in the melting temperature compared with unliganded form and ppGBP-galactose complex. ppGBP binds to monosaccharide, but the structural features revealed it to have an oligosaccharide-binding protein fold, indicating that during evolution the sugar binding pocket may have undergone structural modulation to accommodate monosaccharide only.

  20. Specifications

    International Nuclear Information System (INIS)

    As part of the Danish RERTR Program, three fuel elements with LEU U3O8-Al fuel and three fuel elements with LEU U3Si2-Al fuel were manufactured by NUKEM for irradiation testing in the DR-3 reactor at the Risoe National Laboratory in Denmark. The specifications for the elements with U3O8-Al fuel are presented here as an illustration only. Specifications for the elements with U3Si2-Al fuel were very similar. In this example, materials, material numbers, documents numbers, and drawing numbers specific to a single fabricator have been deleted. (author)

  1. Phylogenetic relationships of the silver saxifrages (Saxifraga, sect. Ligulatae haworth): implications for the evolution of substrate specificity, life histories, and biogeography.

    Science.gov (United States)

    Conti, E; Soltis, D E; Hardig, T M; Schneider, J

    1999-12-01

    The silver saxifrages (Saxifraga sect. Ligulatae Haworth; Saxifragaceae) exhibit remarkable variation of substrate specialization, with strictly calcicole to calcifuge species, as well as life histories which range from semelparity to iteroparity. They occur almost exclusively in the European mountain ranges and display high levels of endemism. Sequences from chloroplast and nuclear ribosomal DNA were obtained to resolve phylogenetic relationships among the silver saxifrages and related taxa and to gain insight into the evolution of substrate specificity, life history, and biogeography. The resulting phylogenies suggested that (1) Saxifraga sect. Ligulatae, as traditionally defined, does not constitute a monophyletic group; (2) lime-secreting hydathodes in calcifuge species apparently represent a secondary nonaptation; (3) semelparity evolved independently two or three times in the silver saxifrages and allied sections, possibly in response to climatic changes that occured during the Pleistocene; and (4) narrow endemics, for example S. cochlearis, likely evolved from the fragmentation of the widespread S. paniculata into refugial populations that became isolated during the glacial maxima of the Pleistocene. PMID:10620412

  2. Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

    Science.gov (United States)

    Ghadikolaei, Kamran Khalili; Shojaei, Maral; Ghaderi, Armin; Hojjati, Farzaneh; Noghabi, Kambiz Akbari; Zahiri, Hossein Shahbani

    2016-08-01

    A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism. PMID:27177969

  3. RNA-Seq approach for genetic improvement of meat quality in pig and evolutionary insight into the substrate specificity of animal carbonyl reductases.

    Directory of Open Access Journals (Sweden)

    Won Yong Jung

    Full Text Available Changes in meat quality traits are strongly associated with alterations in postmortem metabolism which depend on genetic variations, especially nonsynonymous single nucleotide variations (nsSNVs having critical effects on protein structure and function. To selectively identify metabolism-related nsSNVs, next-generation transcriptome sequencing (RNA-Seq was carried out using RNAs from porcine liver, which contains a diverse range of metabolic enzymes. The multiplex SNV genotyping analysis showed that various metabolism-related genes had different nsSNV alleles. Moreover, many nsSNVs were significantly associated with multiple meat quality traits. Particularly, ch7:g.22112616A>G SNV was identified to create a single amino acid change (Thr/Ala at the 145th residue of H1.3-like protein, very close to the putative 147th threonine phosphorylation site, suggesting that the nsSNV may affect multiple meat quality traits by affecting the epigenetic regulation of postmortem metabolism-related gene expression. Besides, one nonsynonymous variation, probably generated by gene duplication, led to a stop signal in porcine testicular carbonyl reductase (PTCR, resulting in a C-terminal (E281-A288 deletion. Molecular docking and energy minimization calculations indicated that the binding affinity of wild-type PTCR to 5α-DHT, a C(21-steroid, was superior to that of C-terminal-deleted PTCR or human carbonyl reductase, which was very consistent with experimental data, reported previously. Furthermore, P284 was identified as an important residue mediating the specific interaction between PTCR and 5α-DHT, and phylogenetic analysis showed that P284 is an evolutionarily conserved residue among animal carbonyl reductases, which suggests that the C-terminal tails of these reductases may have evolved under evolutionary pressure to increase the substrate specificity for C(21-steroids and facilitate metabolic adaptation. Altogether, our RNA-Seq revealed that selective ns

  4. Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

    Directory of Open Access Journals (Sweden)

    Klimacek Mario

    2010-03-01

    Full Text Available Abstract Background In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from Candida tenuis in mutated (NADH-preferring form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate qxylose (g xylose/g dry cell weight/h of 0.08. The study presented herein was performed with the aim of analysing (external factors that limit qxylose of BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose. Results BP10001 and BP000, expressing C. tenuis xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L were converted sequentially, the corresponding qsubstrate values being similar for each strain (glucose: 3.0; xylose: 0.05. The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g as compared

  5. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A. (Sungkyunkwan); (UTSMC)

    2010-08-26

    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  6. Characterization of differences in substrate specificity among CYP1A1, CYP1A2 and CYP1B1: an integrated approach employing molecular docking and molecular dynamics simulations.

    Science.gov (United States)

    Kesharwani, Siddharth S; Nandekar, Prajwal P; Pragyan, Preeti; Rathod, Vijay; Sangamwar, Abhay T

    2016-08-01

    Recent trends in new drug discovery of anticancer drugs have made oncologists more aware of the fact that the new drug discovery must target the developing mechanism of tumorigenesis to improve the therapeutic efficacy of antineoplastic drugs. The drugs designed are expected to have high affinity towards the novel targets selectively. Current research highlights overexpression of CYP450s, particularly cytochrome P450 1A1 (CYP1A1), in tumour cells, representing a novel target for anticancer therapy. However, the CYP1 family is identified as posing significant problems in selectivity of anticancer molecules towards CYP1A1. Three members have been identified in the human CYP1 family: CYP1A1, CYP1A2 and CYP1B1. Although sequences of the three isoform have high sequence identity, they have distinct substrate specificities. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics, design novel antitumour compounds that could be specifically metabolized by only CYP1A1 to mediate their antitumour activity and elucidate the reasons for differences in substrate specificity profile among the three proteins. In the present study, we employed a combination of computational methodologies: molecular docking and molecular dynamics simulations. We utilized eight substrates for elucidating the difference in substrate specificity of the three isoforms. Lastly, we conclude that the substrate specificity of a particular substrate depends upon the type of the active site residues, the dynamic motions in the protein structure upon ligand binding and the physico-chemical characteristics of a particular ligand. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26916064

  7. The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans.

    Science.gov (United States)

    Nisha, M; Satyanarayana, T

    2015-07-01

    In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability.

  8. A novel sucrose hydrolase from the bombycoid silkworms Bombyx mori, Trilocha varians, and Samia cynthia ricini with a substrate specificity for sucrose.

    Science.gov (United States)

    Wang, Huabing; Kiuchi, Takashi; Katsuma, Susumu; Shimada, Toru

    2015-06-01

    Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry.

  9. The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans.

    Science.gov (United States)

    Nisha, M; Satyanarayana, T

    2015-07-01

    In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability. PMID:25573470

  10. Reactor for biological elimination of poorly degradable hydrocarbons by adjustment of substrate-specific retention times; Reaktor zur biologischen Eliminierung schwer abbaubarer Kohlenwasserstoffe durch Einstellung substratspezifischer Verweilzeiten

    Energy Technology Data Exchange (ETDEWEB)

    Brambach, R.

    1997-11-01

    Industrial waste water cleaning increasingly makes use of biological processes based on immobilized biomass, in order to ensure the aimed elimination of toxic substances. A novel, efficient reactor technique was developed, by which dangerous, poorly degradable dissolved hydrocarbons are selectively retained in the bioreactor thanks to an integrated membrane, while other substances are quickly discharged from the system via the effluent. With a synthetic waste water, this selective retention method achieved substrate-specific retention times which were five to fifty times higher than those of a merely hydrodynamically operated reactor system. In addition, the decoupling of high reactor and low effluent concentrations results in a higher biochemical activity in the reactor in comparison with conventional process techniques. (orig.) [Deutsch] In der industriellen Abwasserreinigung werden verstaerkt Bioverfahrenstechniken mit immobilisierter Biomasse eingesetzt, um eine gezielte Eliminierung gefaehrlicher Stoffe durchzufuehren. - Hierzu wurde eine neuartige leistungsfaehige Reaktortechnik entwickelt, die mittels einer im Bioreaktor integrierten Membran gefaehrliche, schwer abbaubare geloeste Kohlenwasserstoffe selektiv im Bioreaktor zurueckhaelt, waehrend andere Stoffe das System schnell ueber den Ablauf verlassen. Durch den stoffselektiven Rueckhalt wurden an einem synthetischen Abwasser substratspezifische Verweilzeiten erzielt, die um den Faktor 5 bis 50 ueber denen eines rein hydrodynamisch bestimmten Reaktorsystems liegen. Darueber hinaus wird aufgrund der Entkopplung von hoher Reaktor- und niedriger Ablaufkonzentration eine im Vergleich zu herkoemmlichen Verfahrenstechniken hoehere biochemische Aktivitaet im Reaktor erzielt. (orig.)

  11. Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

    Science.gov (United States)

    Saleh, Mounir R.

    Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

  12. Validation, automatic generation and use of broad phonetic transcriptions

    NARCIS (Netherlands)

    Bael, Cristophe Patrick Jan Van

    2007-01-01

    Broad phonetic transcriptions represent the pronunciation of words as strings of characters from specifically designed symbol sets. In everyday life, broad phonetic transcriptions are often used as aids to pronounce (foreign) words. In addition, broad phonetic transcriptions are often used for lingu

  13. Dihydrothymidine and thymidine glycol triphosphates as substrates for DNA polymerases: differential recognition of thymine C5-C6 bond saturation and sequence specificity of incorporation.

    OpenAIRE

    Ide, H; Wallace, S. S.

    1988-01-01

    The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated. DHdTTP but not dTTP-GLY was used as a substrate by E. coli DNA polymerase I (Pol I). Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates. The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the mo...

  14. Integrated Broadband Bowtie Antenna on Transparent Silica Substrate

    CERN Document Server

    Zhang, Xingyu; Wang, Shiyi; Subbaraman, Harish; Pan, Zeyu; Zhan, Qiwen; Chen, Ray T

    2016-01-01

    The bowtie antenna is a topic of growing interest in recent years. In this paper, we design, fabricate, and characterize a modified gold bowtie antenna integrated on a transparent silica substrate. The bowtie antenna is designed with broad RF bandwidth to cover the X-band in the electromagnetic spectrum. We numerically investigate the antenna characteristics, specifically its resonant frequency and enhancement factor. Our designed bowtie antenna provides a strong broadband electric field enhancement in its feed gap. Taking advantage of the low-k silica substrate, high enhancement factor can be achieved without the unwanted reflection and scattering from the backside silicon handle which is the issue of using an SOI substrate. We simulate the dependence of resonance frequency on bowtie geometry, and verify the simulation results through experimental investigation, by fabricating different sets of bowtie antennas on silica substrates and then measuring their resonance frequencies. In addition, the far-field rad...

  15. High-dose green tea polyphenol intake decreases CYP3A expression in a liver-specific manner with increases in blood substrate drug concentrations.

    Science.gov (United States)

    Ikarashi, Nobutomo; Ogawa, Sosuke; Hirobe, Ryuta; Kusunoki, Yoshiki; Kon, Risako; Ochiai, Wataru; Sugiyama, Kiyoshi

    2016-06-30

    In recent years, the intake of functional foods containing high-doses of green tea polyphenols (GP) has been increasing. In this study, the long-term safety of high-dose GP was assessed from a pharmacokinetic point of view by focusing on the drug-metabolizing enzyme, cytochrome P450 (CYP). Mice were fed a diet containing 3% GP for 4weeks, and the CYP expression levels and activity were determined. The GP-treated group showed a significant decrease in the hepatic CYP3A and an increase in the hepatic CYP2C expression compared with the control group. CYP1A, CYP2D, and CYP2E expression were not different between the GP-treated and the control groups. In the small intestine, there were no differences in the CYP3A protein levels between the groups. The increase in the plasma triazolam concentration in the GP-treated group was observed. Although no changes were found in the hepatic CYP3A levels in mice receiving a diet containing 0.1% GP for 4weeks, a significant decrease was seen in the hepatic CYP3A level in mice receiving a diet containing 3% GP for only 1week. This study revealed that the intake of a high-dose GP results in a liver-specific decrease in the CYP3A expression level. The results also indicated that the effects of GP on CYP3A were not observed following the intake of a low-dose GP. In the future, caution should be taken in cases when functional foods containing a high-dose GP are concomitantly consumed with a CYP3A substrate drug. PMID:27130545

  16. OXA-46, a new class D beta-lactamase of narrow substrate specificity encoded by a blaVIM-1-containing integron from a Pseudomonas aeruginosa clinical isolate.

    Science.gov (United States)

    Giuliani, Francesco; Docquier, Jean-Denis; Riccio, Maria Letizia; Pagani, Laura; Rossolini, Gian Maria

    2005-05-01

    A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a bla(VIM-1) metallo-beta-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D beta-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of bla(OXA-46) in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D beta-lactamases. PMID:15855521

  17. Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase: possible implications for the HI-6 antidote substrate specificity.

    Science.gov (United States)

    Artursson, Elisabet; Andersson, Per Ola; Akfur, Christine; Linusson, Anna; Börjegren, Susanne; Ekström, Fredrik

    2013-05-01

    Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.

  18. Interleukin 1β Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR.

    Science.gov (United States)

    Shi, Jingxue; He, Yan; Hewett, Sandra J; Hewett, James A

    2016-01-22

    System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

  19. Evaluation of organ-specific glucose metabolism by 18F-FDG in insulin receptor substrate-1 (IRS-1) knockout mice as a model of insulin resistance

    International Nuclear Information System (INIS)

    Insulin resistance (IR) is a physiological condition in which the body produces insulin but does not result in a sufficient biological effect. Insulin resistance is usually asymptomatic but is associated with health problems and is a factor in the metabolic syndrome. The aim of the present study is to clarify organ-specific insulin resistance in normal daily conditions using [18F]-2-fluoro-2-deoxy-D-glucose ([18F]-FDG). The biodistribution of [18F]-FDG was examined in insulin receptor substrate-1 (IRS-1) knockout mice, an animal model of skeletal muscle insulin resistance, and C57BL/6J (wild-type) mice with and without insulin loading. Mice received 0.5 MBq of [18F]-FDG injected into the tail vein, immediately followed by nothing (control cohorts) or an intraperitoneal injection of 1.5 mU/g body weight of human insulin as an insulin loading test. Blood glucose concentrations for all of the experimental animals were assessed at 0, 20, 40, and 60 min post-injection. The mice were subsequently killed, and tissue was collected for evaluation of [18F]-FDG biodistribution. The radioactivity of each organ was measured using a gamma counter. In the absence of insulin, the blood glucose concentrations of wild-type mice (132±26 mg/dl) and IRS-1 knockout mice (134±18 mg/dl) were not significantly different. Blood glucose concentrations decreased following insulin administration, with lower concentrations in wild-type mice than in knockout mice at 20, 40, and 60 min. A statistically significant difference in [18F]-FDG uptake between wild-type mice and IRS-1 knockout mice was confirmed in the heart, abdominal muscle, and femoral muscle. With insulin loading, [18F]-FDG uptake in the heart, back muscle, and abdominal muscle was significantly increased compared to without insulin loading in both wild-type mice and knockout mice. Our results showed that IR significantly affected [18F]-FDG uptake in the heart in normal daily conditions. IR was associated with decreased [18F

  20. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig;

    2007-01-01

    to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has......-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number......Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity...

  1. Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Bak-Jensen, K.S.; André, G.; Gottschalk, T.E.;

    2004-01-01

    in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and activity (k(cat)/K-m) on starch, amylose DP17, and 2-chloro-4-nitrophenyl β-D-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90......% activity, respectively. Thus engineering of aromatic stacking interactions at the ends of the 10-subsite long binding cleft affects activity very differently, dependent on the substrate. Y105A dominates in dual subsite -6/+4 [Y105A/T212(Y/W)] AMY1 mutants having almost retained and low activity on starch......The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites - 6 and +4 ( substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved...

  2. Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme.

    Science.gov (United States)

    Adolph, H W; Maurer, P; Schneider-Bernlöhr, H; Sartorius, C; Zeppezauer, M

    1991-11-01

    1. The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH. 2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E. 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products. 4. The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(II) substitution on the

  3. Germacrene A Synthase in Yarrow (Achillea millefolium Is an Enzyme with Mixed Substrate Specificity: Gene Cloning, Functional Characterization and Expression Analysis

    Directory of Open Access Journals (Sweden)

    Leila ePazouki

    2015-03-01

    Full Text Available Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5 residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS. The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3, functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP, while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP. Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  4. The 1.6 A crystal structure of pyranose dehydrogenase from Agaricus meleagris rationalizes substrate specificity and reveals a flavin intermediate.

    Directory of Open Access Journals (Sweden)

    Tien Chye Tan

    Full Text Available Pyranose dehydrogenases (PDHs are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O activates oxygen by a mechanism that proceeds via a covalent flavin C(4a-hydroperoxide intermediate. Although the flavin C(4a adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2

  5. Specific features of sputtered atoms transport during Ta sub 2 O sub 5 film deposition on substrates of complicated spatial configuration

    CERN Document Server

    Bystrov, Y A; Volpyas, V A; Govako, E A; Timofeev, D E; Troshkov, V V

    2002-01-01

    The possibility of providing for uniform properties of the Ta sub 2 O sub 5 films on the surface of substrates, having complex form, is considered. The attempt is made to consider the sputtered atoms transport in the space of the target-substrate drift through the determination of the thermalization zone geometrical sizes and the subsequent transition of the sputtered particles into the diffusion mode. Various calculational models and static modeling methods are used for describing the process of the sputtered particles transport

  6. Broad Frequency LTCC Vertical Interconnect Transition for Multichip Modules and System on Package Applications

    Science.gov (United States)

    Decrossas, Emmanuel; Glover, Michael D.; Porter, Kaoru; Cannon, Tom; Mantooth, H. Alan; Hamilton, M. C.

    2013-01-01

    Various stripline structures and flip chip interconnect designs for high-speed digital communication systems implemented in low temperature co-fired ceramic (LTCC) substrates are studied in this paper. Specifically, two different transition designs from edge launch 2.4 millimeter connectors to stripline transmission lines embedded in LTCC are discussed. After characterizing the DuPont (sup trademark) 9K7 green tape, different designs are proposed to improve signal integrity for high-speed digital data. The full-wave simulations and experimental data validate the presented designs over a broad frequency band from Direct Current to 50 gigahertz and beyond.

  7. Integrating Hot and Cool Intelligences: Thinking Broadly about Broad Abilities

    Directory of Open Access Journals (Sweden)

    W. Joel Schneider

    2016-01-01

    Full Text Available Although results from factor-analytic studies of the broad, second-stratum abilities of human intelligence have been fairly consistent for decades, the list of broad abilities is far from complete, much less understood. We propose criteria by which the list of broad abilities could be amended and envision alternatives for how our understanding of the hot intelligences (abilities involving emotionally-salient information and cool intelligences (abilities involving perceptual processing and logical reasoning might be integrated into a coherent theoretical framework.

  8. Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3

    DEFF Research Database (Denmark)

    Wandall, H H; Hassan, H; Mirgorodskaya, E;

    1997-01-01

    Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified...... recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3......, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem...

  9. Purification and substrate specificity of beta-xylosidase from sycamore cell (Acer pseudoplatanus L.): application for structural analysis of xylose-containing N-linked oligosaccharides.

    Science.gov (United States)

    Tezuka, K; Hayashi, M; Ishihara, H; Nishimura, M; Onozaki, K; Takahashi, N

    1993-06-01

    A beta-xylosidase was purified 51-fold from culture medium of sycamore (Acer pseudoplatanus L.) cells using p-nitrophenyl beta-D-xylopyranoside as a substrate. This enzyme can remove a xylose residue from asparagine-linked oligosaccharides, derivatized with 2-aminopyridine. A pentasaccharide, Xy1 beta 2Man beta 4GlcNAc beta 4(Fuc-alpha 3)GlcNAc was the favorite substrate in N-linked oligosaccharides, but a xylose residue in Xy1 beta 2(Man-alpha 3)Man beta sequence could not be removed by the enzyme. We also propose an efficient method for detection of xylose residue in N-linked oligosaccharides by a combination of the two-dimensional sugar mapping technique and the xylosidase digestion.

  10. Measurement of Acylcarnitine Substrate to Product Ratios Specific to Biotin-Dependent Carboxylases Offers a Combination of Indicators of Biotin Status in Humans12

    OpenAIRE

    Bogusiewicz, Anna; Horvath, Thomas D; Stratton, Shawna L.; Mock, Donald M; Boysen, Gunnar

    2012-01-01

    This work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine : 3-methylglutarylcarnitine (3HIAc : MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio of propionylc...

  11. Offshore Substrate

    Data.gov (United States)

    California Department of Resources — This shapefile displays the distribution of substrate types from Pt. Arena to Pt. Sal in central/northern California. Originally this data consisted of seven paper...

  12. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra

    2014-03-12

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  13. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Science.gov (United States)

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  14. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sandra Witz

    Full Text Available Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  15. Multiple alternative substrate kinetics.

    Science.gov (United States)

    Anderson, Vernon E

    2015-11-01

    The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the "specificity constant" (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment.

  16. Effects Of Substrate Scattering On Bar-Code Scanning Signals

    Science.gov (United States)

    Barkan, Eric; Sklar, David

    1983-04-01

    When a beam of light strikes a piece of paper or similar substrate, a portion of its energy penetrates into the bulk and, due to multiple scattering, may re-emerge at some distance from the point of entry. We refer to this phenomenon as substrate scattering. In this paper we describe a general model for scattering substrates and, using linear systems theory, we investigate its implications for bar-code scanning. We show that the effects of substrate scattering can be represented as a modified reflectance distribution associated with the original printed reflectance pattern. This effective distribution is shown to be independent of the details of scanning system configuration. We show that under a broad range of conditions substrate scattering will decrease modulation and will cause scanners to overestimate barwidths. A specific diffusion model of the scattering process is developed, providing a family of functions which can be used in empirical studies. We conclude that the scattering distributions will not even be approximately Gaussian. Experimental results are presented which are consistent with this conclusion and inconsistent with a Gaussian model. Further experimental results are presented which show that, for typical substrates, depth of modulation may be decreased by 20 percent or more and perceived bar-width increases will be on the order of one mil.

  17. Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.).

    Science.gov (United States)

    Tezuka, K; Hayashi, M; Ishihara, H; Akazawa, T; Takahashi, N

    1992-02-01

    We measured the activities of alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, beta-1,4-mannosyl-glycoprotein beta-1,2-xylosyltransferase and glycoprotein 3-alpha-L-fucosyltransferase in the Golgi fraction of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence-labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two-dimensional sugar mapping using high-performance liquid chromatography. We demonstrated that (formula; see text) was processed to produce by these in vitro reactions. On the basis of these results, we discuss a biosynthetic pathway for xylose containing N-linked oligosaccharides in plant glycoproteins.

  18. FANCJ helicase uniquely senses oxidative base damage in either strand of duplex DNA and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner.

    Science.gov (United States)

    Suhasini, Avvaru N; Sommers, Joshua A; Mason, Aaron C; Voloshin, Oleg N; Camerini-Otero, R Daniel; Wold, Marc S; Brosh, Robert M

    2009-07-01

    FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism. PMID:19419957

  19. The Broad Autism Phenotype Questionnaire

    Science.gov (United States)

    Hurley, Robert S. E.; Losh, Molly; Parlier, Morgan; Reznick, J. Steven; Piven, Joseph

    2007-01-01

    The broad autism phenotype (BAP) is a set of personality and language characteristics that reflect the phenotypic expression of the genetic liability to autism, in non-autistic relatives of autistic individuals. These characteristics are milder but qualitatively similar to the defining features of autism. A new instrument designed to measure the…

  20. Substrate Handbook for Biogas Production; Substrathandbok foer biogasproduktion

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, My; Uldal, Martina (AnoxKaldnes AB, Lund (Sweden))

    2009-02-15

    Today, co-digestion plants in Sweden treat a broad range of different substrates, of which some have not previously been used for anaerobic digestion. The major part of this organic waste derives from households, restaurants, food industries and farms. When evaluating a new substrate as feed for anaerobic digestion, several different aspects need to be taken into consideration, such as anaerobic degradability, TS/VS content, nutrient composition and risk for mechanical problems. Consequently, there is a need for practical guidelines on how to evaluate new substrates as raw materials for biogas production, including not only gas yield but also what practical and microbiological problems that may arise when the specific substrate is treated together with other substrates in the plant. The aim with this handbook is to provide a basis on how to evaluate new substrates as feed for anaerobic digestion. The intention is that this material will save time and effort for the personnel at the plant when they come in contact with new types of waste. Also, the aim is to facilitate the process of identifying new substrates within the ABP-regulation (1774/2002) and what requirements are then demanded on handling. The work with the handbook has been divided in three different parts; (1) an extensive literature study and a compilation of the achieved results, (2) interviews with personnel at most of the Swedish co-digestion plants to identify substrates and problems of interest, and (3) lab tests of selected substrates. The lab tests included Bio Methane Potential (BMP) tests as well as a simple characterization of each substrate based on fat/protein/carbohydrate content. All data origins from anaerobic digestion within the mesophilic temperature range, but the results and discussion are applicable also for thermophilic anaerobic digestion. The result of this work is a written report together with an Excel file which are to be directly used by the biogas plants as a basis in the

  1. Structural evidence of substrate specificity in mammalian peroxidases: structure of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 A resolution.

    Science.gov (United States)

    Sheikh, Ishfaq Ahmed; Singh, Amit Kumar; Singh, Nagendra; Sinha, Mau; Singh, S Baskar; Bhushan, Asha; Kaur, Punit; Srinivasan, Alagiri; Sharma, Sujata; Singh, Tej P

    2009-05-29

    The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN(-)) has been determined at 2.4A resolution. It revealed that the SCN(-) ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN(-) ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN(-) forms a hydrogen bond with a water (Wat) molecule at position 6'. This water molecule is stabilized by two hydrogen bonds with Gln(423) N(epsilon2) and Phe(422) oxygen. In contrast, the placement of the SCN(-) ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln(423), Phe(422) oxygen, and Wat(6)' in LPO is occupied primarily by the side chain of Phe(407) in MPO due to an entirely different conformation of the loop corresponding to the segment Arg(418)-Phe(431) of LPO. This arrangement in MPO does not favor a similar orientation of the SCN(-) ion. The orientation of the catalytic product OSCN(-) as reported in the structure of LPO.OSCN(-) is similar to the orientation of SCN(-) in the structure of LPO.SCN(-). Similarly, in the structure of LPO.SCN(-).CN(-), in which CN(-) binds at Wat(1), the position and orientation of the SCN(-) ion are also identical to that observed in the structure of LPO.SCN.

  2. Broad Diphotons from Narrow States

    CERN Document Server

    An, Haipeng; Zhang, Yue

    2015-01-01

    ATLAS and CMS have each reported a modest diphoton excess consistent with the decay of a broad resonance at ~ 750 GeV. We show how this signal can arise in a weakly coupled theory comprised solely of narrow width particles. In particular, if the decaying particle is produced off-shell, then the associated diphoton resonance will have a broad, adjustable width. We present simplified models which explain the diphoton excess through the three-body decay of a scalar or fermion. Our minimal ultraviolet completion is a weakly coupled and renormalizable theory of a singlet scalar plus a heavy vector-like quark and lepton. The smoking gun of this mechanism is an asymmetric diphoton peak recoiling against missing transverse energy, jets, or leptons.

  3. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.

    Science.gov (United States)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik

    2007-04-01

    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

  4. Cochlear microphonic broad tuning curves

    Science.gov (United States)

    Ayat, Mohammad; Teal, Paul D.; Searchfield, Grant D.; Razali, Najwani

    2015-12-01

    It is known that the cochlear microphonic voltage exhibits much broader tuning than does the basilar membrane motion. The most commonly used explanation for this is that when an electrode is inserted at a particular point inside the scala media, the microphonic potentials of neighbouring hair cells have different phases, leading to cancelation at the electrodes location. In situ recording of functioning outer hair cells (OHCs) for investigating this hypothesis is exceptionally difficult. Therefore, to investigate the discrepancy between the tuning curves of the basilar membrane and those of the cochlear microphonic, and the effect of phase cancellation of adjacent hair cells on the broadness of the cochlear microphonic tuning curves, we use an electromechanical model of the cochlea to devise an experiment. We explore the effect of adjacent hair cells (i.e., longitudinal phase cancellation) on the broadness of the cochlear microphonic tuning curves in different locations. The results of the experiment indicate that active longitudinal coupling (i.e., coupling with active adjacent outer hair cells) only slightly changes the broadness of the CM tuning curves. The results also demonstrate that there is a π phase difference between the potentials produced by the hair bundle and the soma near the place associated with the characteristic frequency based on place-frequency maps (i.e., the best place). We suggest that the transversal phase cancellation (caused by the phase difference between the hair bundle and the soma) plays a far more important role than longitudinal phase cancellation in the broadness of the cochlear microphonic tuning curves. Moreover, by increasing the modelled longitudinal resistance resulting the cochlear microphonic curves exhibiting sharper tuning. The results of the simulations suggest that the passive network of the organ of Corti determines the phase difference between the hair bundle and soma, and hence determines the sharpness of the

  5. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  6. A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification, substrate specificity, and expression in Salmonella waaC mutants.

    Science.gov (United States)

    Kanipes, Margaret I; Ribeiro, Anthony A; Lin, Shanhua; Cotter, Robert J; Raetz, Christian R H

    2003-05-01

    The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]lipid IV(A) as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-[4'-(32)P]lipid IV(A). A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A). The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A). Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.

  7. Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure*

    OpenAIRE

    Awai, Takako; Ochi, Anna; Ihsanawati,; Sengoku, Toru; Hirata, Akira; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Hori, Hiroyuki

    2011-01-01

    Archaeal and eukaryotic tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) produces N2,N2-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Tr...

  8. Host plant-dependent metabolism of 4-hydroxybenzylglucosinolate in Pieris rapae: substrate specificity and effects of genetic modification and plant nitrile hydratase.

    Science.gov (United States)

    Agerbirk, Niels; Olsen, Carl Erik; Topbjerg, Henrik Bak; Sørensen, Jens Christian

    2007-11-01

    After ingestion of transgenic Arabidopsis thaliana CYP79A1 containing sinalbin (4-hydroxybenzylglucosinolate) due to genetic modification, only one major sinalbin-derived sulphate ester (the sulphate ester of 4-hydroxyphenylacetonitrile) was excreted by Pieris rapae caterpillars (corresponding to 69mol% of ingested sinalbin). An additional sulphate ester (the sulphate ester of 4-hydroxyphenylacetamide) was excreted when the caterpillars were reared on two plant species (Sinapis alba and Sinapis arvensis) that contained sinalbin naturally. Artificial addition of sinalbin to S. arvensis leaves resulted in increased levels of the sulphated amide, and an enzymatic activity (nitrile hydratase) explaining the formation of the sulphated amide from sinalbin was detected in both Sinapis species, but not in A. thaliana. In agreement with the suggested minor metabolic pathway, the caterpillars were able to sulphate 4-hydroxyphenylacetamide offered as part of an artificial diet. In fact, phenol and seven para-substituted phenol derivatives with substituents of moderate size were sulphated and excreted, but all tested phenols devoid of a nitrile functional group were less efficiently sulphated than the primary sinalbin detoxification product, 4-hydroxyphenylacetonitrile. This suggests that the specificity of the sulphation step involved in sinalbin metabolism may be adapted to nitriles formed as metabolites of phenolic glucosinolates. On the contrary, there was no specificity for products (4-hydroxybenzylascorbigen and 4-hydroxybenzylalcohol) derived from the semistable isothiocyanate produced from sinalbin in the absence of nitrile specifier protein. PMID:17916498

  9. Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

    Directory of Open Access Journals (Sweden)

    Lara Wohlbold

    2012-08-01

    Full Text Available The cyclin-dependent kinases (CDKs that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS Cdk2 after exposure to ionizing radiation (IR enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

  10. Substrate tolerant direct block copolymer nanolithography

    DEFF Research Database (Denmark)

    Li, Tao; Wang, Zhongli; Schulte, Lars;

    2016-01-01

    Block copolymer (BC) self-assembly constitutes a powerful platform for nanolithography. However, there is a need for a general approach to BC lithography that critically considers all the steps from substrate preparation to the final pattern transfer. We present a procedure that significantly sim...... plasma treatment enables formation of the oxidized PDMS hard mask, PS block removal and polymer or graphene substrate patterning....... simplifies the main stream BC lithography process, showing a broad substrate tolerance and allowing for efficient pattern transfer over wafer scale. PDMS-rich poly(styrene-b-dimethylsiloxane) (PS-b-PDMS) copolymers are directly applied on substrates including polymers, silicon and graphene. A single oxygen...

  11. Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection.

    Science.gov (United States)

    Havens, J A; Etges, W J

    2013-03-01

    Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within-population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair-mated adults from no-choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population-specific plasticity in CHCs. Different groups of CHCs mediated female choice-based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems.

  12. Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction.

    Directory of Open Access Journals (Sweden)

    Yunguang Wang

    Full Text Available The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes' expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.

  13. Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase.

    Science.gov (United States)

    Price, Claire L; Warrilow, Andrew G S; Parker, Josie E; Mullins, Jonathan G L; Nes, W David; Kelly, Diane E; Kelly, Steven L

    2015-05-15

    Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide. PMID:25746994

  14. S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids.

    Science.gov (United States)

    Alves, L C; Melo, R L; Sanderson, S J; Mottram, J C; Coombs, G H; Caliendo, G; Santagada, V; Juliano, L; Juliano, M A

    2001-03-01

    We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and

  15. Ghost imaging with broad distance

    Institute of Scientific and Technical Information of China (English)

    段德洋; 张路; 杜少将; 夏云杰

    2015-01-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is.

  16. Ghost imaging with broad distance

    Science.gov (United States)

    Duan, De-Yang; Zhang, Lu; Du, Shao-Jiang; Xia, Yun-Jie

    2015-10-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is. Project supported by the National Natural Science Foundation of China (Grant Nos. 61178012, 11204156, 11304179, and 11247240), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant Nos. 20133705110001 and 20123705120002), the Scientific Research Foundation for Outstanding Young Scientists of Shandong Province, China (Grant No. BS2013DX034), and the Natural Science Foundation of Shandong Province, China (Grant No. ZR2012FQ024).

  17. Prebiotics: preferential substrates for specific germs?

    Science.gov (United States)

    Roberfroid, M B

    2001-02-01

    A prebiotic is "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon." The premise is based on the hypothesis that the large gut in humans contains bacteria that are beneficial or detrimental to health. Although this generalization probably gives too simplistic a view of gut microbiology, it is a feasible working concept. Currently, food components that seem to exert the best prebiotic effects are inulin-type fructans. In pure culture, most species of bifidobacteria are adapted to the utilization of these nondigestible oligosaccharides but many other bacteria are also capable of metabolizing them. Clearly, these studies of pure bacteria are of limited use unless their results are supported by the results of studies using mixed cultures. Indeed, as many components of the gut microbiota as possible should be measured to indicate a true prebiotic effect. Simple stimulation of bifidobacteria is insufficient to demonstrate an effect; the effects on other gut microorganisms in vivo with human volunteers is necessary. Adjustment of the composition and activities of the colonic microflora so that health-promoting activities are optimized remains key in functional food development. New methods are being applied extensively to human gut microbiology and promise the degree of reliability required to detect subtle changes in colonic microflora composition and to correlate such changes with health benefits. This is a review of the present state of knowledge concerning prebiotics, with emphasis on the criteria used for classification, mechanisms of selective growth stimulation, and physiologic effects.

  18. 78 FR 20119 - Broad Stakeholder Survey

    Science.gov (United States)

    2013-04-03

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 30-day... soliciting comments concerning the Broad Stakeholder Survey. DHS previously published this ICR in the Federal... responders across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  19. 77 FR 50144 - Broad Stakeholder Survey

    Science.gov (United States)

    2012-08-20

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until... across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  20. 76 FR 34087 - Broad Stakeholder Survey

    Science.gov (United States)

    2011-06-10

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until.... The Broad Stakeholder Survey is designed to gather stakeholder feedback on the effectiveness of...

  1. Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

    Directory of Open Access Journals (Sweden)

    Nathan E Kreel

    Full Text Available Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363 found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

  2. Study of Carbon Nanotube-Substrate Interaction

    Directory of Open Access Journals (Sweden)

    Jaqueline S. Soares

    2012-01-01

    Full Text Available Environmental effects are very important in nanoscience and nanotechnology. This work reviews the importance of the substrate in single-wall carbon nanotube properties. Contact with a substrate can modify the nanotube properties, and such interactions have been broadly studied as either a negative aspect or a solution for developing carbon nanotube-based nanotechnologies. This paper discusses both theoretical and experimental studies where the interaction between the carbon nanotubes and the substrate affects the structural, electronic, and vibrational properties of the tubes.

  3. Power electronics substrate for direct substrate cooling

    Science.gov (United States)

    Le, Khiet; Ward, Terence G.; Mann, Brooks S.; Yankoski, Edward P.; Smith, Gregory S.

    2012-05-01

    Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

  4. Physical masking process for integrating micro metallic structures on polymer substrate

    DEFF Research Database (Denmark)

    Islam, Mohammad Aminul; Hansen, Hans Nørgaard

    2009-01-01

    plasmon devices need micro metallic structures on a polymer substrate with an uniform metal layer thickness in the nanometer range. A well known fabrication process to achieve such metallic surface pattern on polymer substrate is photolithography which involves an expensive mask and toxic chemicals....... The current study shows a novel approach for fabricating thin micro metallic structures on polymer substrates using a simple physical mask and a PVD equipment. The new process involves fewer process steps, it is cost effective and suitable for high volume industrial production. Current study suggests......Integration of micro metallic structures in polymer devices is a broad multi-disciplinary research field, consisting of various combinations of mechanical, chemical and physical fabrication methods. Each of the methods has its specific advantages and disadvantages. Some applications like surface...

  5. Broadband antireflection nanodome structures on SiC substrate

    DEFF Research Database (Denmark)

    Ou, Yiyu; Zhu, Xiaolong; Møller, Uffe Visbech;

    2013-01-01

    Nanodome structures are demonstrated on the SiC substrate by using nanosphere lithography and dry etching. Significant surface antireflection has been observed over a broad spectral range from 400 nm to 1600 nm.......Nanodome structures are demonstrated on the SiC substrate by using nanosphere lithography and dry etching. Significant surface antireflection has been observed over a broad spectral range from 400 nm to 1600 nm....

  6. Substrate flexibility of vicenisaminyltransferase VinC involved in the biosynthesis of vicenistatin.

    Science.gov (United States)

    Minami, Atsushi; Eguchi, Tadashi

    2007-04-25

    A glycosyltransferase VinC is involved in the biosynthesis of antitumor beta-glycoside antibiotic vicenistatin. It catalyzes a glycosyl transfer reaction between dTDP-alpha-D-vicenisamine and vicenilactam. Previous identification of its broad substrate specificity toward various glycosyl acceptors enabled us to explore the potential of VinC for glycodiversification. In vitro study of the substrate specificity toward several dTDP-sugars with vicenilactam established that VinC displayed activities with alpha-anomers of several dTDP-2-deoxy-D-sugars such as mycarose, digitoxose, olivose, and 2-deoxyglucose to afford respective beta-glycosides. Notably, beta-anomers of dTDP-2-deoxy-D-sugars also appeared to be accepted by VinC to form alpha-glycosides. Furthermore, VinC is capable of catalyzing glycosyl transfer reactions from both the alpha-anomer and beta-anomer of dTDP-l-mycarose, respectively, into beta-glycoside and alpha-glycoside. These results indicate that VinC is a unique glycosyltransferase possessing broad substrate specificity. The mechanism of this axially oriented glycosidic bond formation from the equatorially oriented dTDP-sugar might be explained by conformational change of dTDP-sugar to a boat conformation during the glycosyl transfer reaction. To apply these features of VinC for glycodiversification, 22 sets of structurally diverse glycosides were constructed using unnatural glycosyl donors and acceptors. PMID:17388594

  7. Broad Prize: Do the Successes Spread?

    Science.gov (United States)

    Samuels, Christina A.

    2011-01-01

    When the Broad Prize for Urban Education was created in 2002, billionaire philanthropist Eli Broad said he hoped the awards, in addition to rewarding high-performing school districts, would foster healthy competition; boost the prestige of urban education, long viewed as dysfunctional; and showcase best practices. Over the 10 years the prize has…

  8. Direct transfer of graphene onto flexible substrates

    Science.gov (United States)

    Pimenta, Luiz Gustavo; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred; Kong, Jing; Araujo, Paulo

    2014-03-01

    We explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate. Various substrates of general interest in research and industry were studied including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate's hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate present processes and open up directions for applications of chemical vapor deposition (CVD) graphene on flexible substrates. A broad range of applications of CVD graphene can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene.

  9. Measuring Prevention More Broadly, An Empirical...

    Data.gov (United States)

    U.S. Department of Health & Human Services — Measuring Prevention More Broadly, An Empirical Assessment of CHIPRA Core Measures Differences in CHIP design and structure, across states and over time, may limit...

  10. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    Science.gov (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models. PMID:27415450

  11. Capturing a Dynamic Chaperone-Substrate Interaction Using NMR-Informed Molecular Modeling.

    Science.gov (United States)

    Salmon, Loïc; Ahlstrom, Logan S; Horowitz, Scott; Dickson, Alex; Brooks, Charles L; Bardwell, James C A

    2016-08-10

    Chaperones maintain a healthy proteome by preventing aggregation and by aiding in protein folding. Precisely how chaperones influence the conformational properties of their substrates, however, remains unclear. To achieve a detailed description of dynamic chaperone-substrate interactions, we fused site-specific NMR information with coarse-grained simulations. Our model system is the binding and folding of a chaperone substrate, immunity protein 7 (Im7), with the chaperone Spy. We first used an automated procedure in which NMR chemical shifts inform the construction of system-specific force fields that describe each partner individually. The models of the two binding partners are then combined to perform simulations on the chaperone-substrate complex. The binding simulations show excellent agreement with experimental data from multiple biophysical measurements. Upon binding, Im7 interacts with a mixture of hydrophobic and hydrophilic residues on Spy's surface, causing conformational exchange within Im7 to slow down as Im7 folds. Meanwhile, the motion of Spy's flexible loop region increases, allowing for better interaction with different substrate conformations, and helping offset losses in Im7 conformational dynamics that occur upon binding and folding. Spy then preferentially releases Im7 into a well-folded state. Our strategy has enabled a residue-level description of a dynamic chaperone-substrate interaction, improving our understanding of how chaperones facilitate substrate folding. More broadly, we validate our approach using two other binding partners, showing that this approach provides a general platform from which to investigate other flexible biomolecular complexes through the integration of NMR data with efficient computational models.

  12. Personal glucose meters for detection and quantification of a broad range of analytes

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Yi; Xiang, Yu

    2015-02-03

    A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

  13. Mapping plant functional types over broad mountainous regions

    OpenAIRE

    Danlu Cai; Yanning Guan; Shan Guo; Chunyan Zhang; Klaus Fraedrich

    2014-01-01

    Research on global climate change requires plant functional type (PFT) products. Although several PFT mapping procedures for remote sensing imagery are being used, none of them appears to be specifically designed to map and evaluate PFTs over broad mountainous areas which are highly relevant regions to identify and analyze the response of natural ecosystems. We present a methodology for generating soft classifications of PFTs from remotely sensed time series that are based on a hierarchical s...

  14. BROAD PHONEME CLASSIFICATION USING SIGNAL BASED FEATURES

    Directory of Open Access Journals (Sweden)

    Deekshitha G

    2014-12-01

    Full Text Available Speech is the most efficient and popular means of human communication Speech is produced as a sequence of phonemes. Phoneme recognition is the first step performed by automatic speech recognition system. The state-of-the-art recognizers use mel-frequency cepstral coefficients (MFCC features derived through short time analysis, for which the recognition accuracy is limited. Instead of this, here broad phoneme classification is achieved using features derived directly from the speech at the signal level itself. Broad phoneme classes include vowels, nasals, fricatives, stops, approximants and silence. The features identified useful for broad phoneme classification are voiced/unvoiced decision, zero crossing rate (ZCR, short time energy, most dominant frequency, energy in most dominant frequency, spectral flatness measure and first three formants. Features derived from short time frames of training speech are used to train a multilayer feedforward neural network based classifier with manually marked class label as output and classification accuracy is then tested. Later this broad phoneme classifier is used for broad syllable structure prediction which is useful for applications such as automatic speech recognition and automatic language identification.

  15. Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

    Science.gov (United States)

    Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

    2016-08-25

    Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. PMID:27565346

  16. Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.

    Science.gov (United States)

    Dang, Michelle; Armbruster, Nicole; Miller, Miles A; Cermeno, Efrain; Hartmann, Monika; Bell, George W; Root, David E; Lauffenburger, Douglas A; Lodish, Harvey F; Herrlich, Andreas

    2013-06-11

    Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.

  17. Carbohydrate utilization patterns for the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus reveal broad growth substrate preferences

    NARCIS (Netherlands)

    Vanfossen, A.L.; Verhaart, M.R.A.; Kengen, S.W.M.; Kelly, R.M.

    2009-01-01

    Co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on indiv

  18. Giant Broad Line Regions in Dwarf Seyferts

    CERN Document Server

    Devereux, Nick

    2015-01-01

    High angular resolution spectroscopy obtained with the Hubble Space Telescope (HST) has revealed a remarkable population of galaxies hosting dwarf Seyfert nuclei with an unusually large broad-line region (BLR). These objects are remarkable for two reasons. Firstly, the size of the BLR can, in some cases, rival those seen in the most luminous quasars. Secondly, the size of the BLR is not correlated with the central continuum luminosity, an observation that distinguishes them from their reverberating counterparts. Collectively, these early results suggest that non-reverberating dwarf Seyferts are a heterogeneous group and not simply scaled versions of each other. Careful inspection reveals broad H Balmer emission lines with single peaks, double peaks, and a combination of the two, suggesting that the broad emission lines are produced in kinematically distinct regions centered on the black hole (BH). Because the gravitational field strength is already known for these objects, by virtue of knowing their BH mass, ...

  19. Silicon micromachined broad band light source

    Science.gov (United States)

    George, Thomas (Inventor); Jones, Eric (Inventor); Tuma, Margaret L. (Inventor); Eastwood, Michael (Inventor); Hansler, Richard (Inventor)

    2004-01-01

    A micro electromechanical system (MEMS) broad band incandescent light source includes three layers: a top transmission window layer; a middle filament mount layer; and a bottom reflector layer. A tungsten filament with a spiral geometry is positioned over a hole in the middle layer. A portion of the broad band light from the heated filament is reflective off the bottom layer. Light from the filament and the reflected light of the filament are transmitted through the transmission window. The light source may operate at temperatures of 2500 K or above. The light source may be incorporated into an on board calibrator (OBC) for a spectrometer.

  20. Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis.

    Science.gov (United States)

    Nasrollahi, Samira; Golalizadeh, Leila; Sajedi, Reza H; Taghdir, Majid; Asghari, S Mohsen; Rassa, Mehdi

    2013-09-01

    The gene encoding a maltogenic amylase (MAase) from a newly isolated strain of thermophilic Geobacillus has been isolated, cloned and expressed. Following purification, biochemical and structural characterization have been performed. The enzyme exhibited maximal activity at a broad temperature range between 55 and 65 °C, clearly higher than that of other dimeric MAses. The optimum pH was 6.0 and catalytic activity increased by of Li(+) and K(+). A noticeable preference was demonstrated for α-, β- and γ-cyclodextrin (CD) compared to polymeric substrates (amylose, amylopectin, glycogen and starch) possibly due to steric interference. The affinity for CD substrates increased in the order of γ-CD>β-CD>α-CD, but k(cat)/K(m) increased as α-CD>β-CD>γ-CD, implying that increased substrate specificities are mainly attribute to kcat. Thermodynamic analysis of the activation process showed that improved activity (decrease in ΔG(#)) is accompanied by increases in activation entropy (ΔS(#)) for aforementioned substrates. Molecular docking on the binding interactions between substrates and active site residues revealed a considerably higher accessible surface area for the active site residues in the presence of α-CD than β-CD, indicating that interactions in the transition state are stronger in the presence of α-CD. This result explains the increased ΔH(#) of the activation process and increased K(m) of the enzyme in the presence of α-CD, compared to that of β-CD. This study, which presents the first detailed comparative analysis on the substrate preference of dimeric MAases for different substrates, may shed some lights into the molecular mechanism of these enzymes.

  1. The GREGOR Broad-Band Imager

    Science.gov (United States)

    von der Lühe, O.; Volkmer, R.; Kentischer, T. J.; Geißler, R.

    2012-11-01

    The design and characteristics of the Broad-Band Imager (BBI) of GREGOR are described. BBI covers the visible spectral range with two cameras simultaneously for a large field and with critical sampling at 390 nm, and it includes a mode for observing the pupil in a Foucault configuration. Samples of first-light observations are shown.

  2. Broad resonances and beta-decay

    DEFF Research Database (Denmark)

    Riisager, K.; Fynbo, H. O. U.; Hyldegaard, S.;

    2015-01-01

    Beta-decay into broad resonances gives a distorted lineshape in the observed energy spectrum. Part of the distortion arises from the phase space factor, but we show that the beta-decay matrix element may also contribute. Based on a schematic model for p-wave continuum neutron states it is argued...

  3. Broad iron lines in Active Galactic Nuclei

    CERN Document Server

    Fabian, A C; Reynolds, C S; Young, A J

    2000-01-01

    An intrinsically narrow line emitted by an accretion disk around a black hole appears broadened and skewed as a result of the Doppler effect and gravitational redshift. The fluorescent iron line in the X-ray band at 6.4-6.9keV is the strongest such line and is seen in the X-ray spectrum of many active galactic nuclei and, in particular, Seyfert galaxies. It is an important diagnostic with which to study the geometry and other properties of the accretion flow very close to the central black hole. The broad iron line indicates the presence of a standard thin accretion disk in those objects, often seen at low inclination. The broad iron line has opened up strong gravitational effects around black holes to observational study with wide-reaching consequences for both astrophysics and physics.

  4. Broad line regions in Seyfert-1 galaxies

    International Nuclear Information System (INIS)

    To reproduce observed emission profiles of Seyfert galaxies, rotation in an accretion disk has been proposed. In this thesis, the profiles emitted by such an accretion disk are investigated. Detailed comparison with the observed profiles yields that a considerable fraction can be fitted with a power-law function, as predicted by the model. The author analyzes a series of high quality spectra of Seyfert galaxies, obtained with the 2.5m telescope at Las Campanas. He presents detailed analyses of two objects: Mkn335 and Akn120. In both cases, strong evidence is presented for the presence of two separate broad line zones. These zones are identified with an accretion disk and an outflowing wind. The disk contains gas with very high densities and emits predominantly the lower ionization lines. He reports on the discovery of very broad wings beneath the strong forbidden line 5007. (Auth.)

  5. Fourier evaluation of broad Moessbauer spectra

    International Nuclear Information System (INIS)

    It is shown by the Fourier analysis of broad Moessbauer spectra that the even part of the distribution of the dominant hyperfine interaction (hyperfine field or quadrupole splitting) can be obtained directly without using least-square fitting procedures. Also the odd part of this distribution correlated with other hyperfine parameters (e.g. isomer shift) can be directly determined. Examples for amorphous magnetic and paramagnetic iron-based alloys are presented. (author)

  6. Crx broadly modulates the pineal transcriptome

    OpenAIRE

    Rovsing, Louise; Clokie, Samuel; Bustos, Diego M.; Rohde, Kristian; Steven L Coon; Litman, Thomas; Rath, Martin F.; Møller, Morten; Klein, David C.

    2011-01-01

    Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. Here, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse p...

  7. A Broad View of Macroeconomic Stability

    OpenAIRE

    José Antonio Ocampo

    2005-01-01

    This paper recommends a broad concept of macroeconomic stability, whereby “sound macroeconomic frameworks” include not only price stability and sound fiscal policies, but also a well-functioning real economy, sustainable debt ratios and healthy public and private sector balance sheets. These multiple dimensions imply using multiple policy instruments. The paper elaborates a framework for developing countries that involves active use of counter-cyclical macroeconomic policies (exchange rate, m...

  8. Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals.

    Science.gov (United States)

    Caputo, Alessandro T; Alonzi, Dominic S; Marti, Lucia; Reca, Ida-Barbara; Kiappes, J L; Struwe, Weston B; Cross, Alice; Basu, Souradeep; Lowe, Edward D; Darlot, Benoit; Santino, Angelo; Roversi, Pietro; Zitzmann, Nicole

    2016-08-01

    The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design. PMID:27462106

  9. Relativistic redshifts in quasar broad lines

    CERN Document Server

    Tremaine, Scott; Liu, Xin; Loeb, Abraham

    2014-01-01

    The broad emission lines commonly seen in quasar spectra have velocity widths of a few per cent of the speed of light, so special- and general-relativistic effects have a significant influence on the line profile. We have determined the redshift of the broad H-beta line in the quasar rest frame (determined from the core component of the [OIII] line) for over 20,000 quasars from the Sloan Digital Sky Survey DR7 quasar catalog. The mean redshift as a function of line width is approximately consistent with the relativistic redshift that is expected if the line originates in a randomly oriented Keplerian disk that is obscured when the inclination of the disk to the line of sight exceeds ~30-45 degrees, consistent with simple AGN unification schemes. This result also implies that the net line-of-sight inflow/outflow velocities in the broad-line region are much less than the Keplerian velocity when averaged over a large sample of quasars with a given line width.

  10. Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells.

    Science.gov (United States)

    Kourouklis, Andreas P; Kaylan, Kerim B; Underhill, Gregory H

    2016-08-01

    Recent approaches have utilized microfabricated platforms to examine combinations of microenvironmental signals that regulate stem and progenitor cell differentiation. However, the majority of these efforts have focused on the biochemical properties of extracellular matrix (ECM) or soluble factors without simultaneously exploring the biomechanical effects of cell-substrate interactions. To address this need, we combined a high-throughput approach for the analysis of combinatorial ECM cues with substrates of modular stiffness and traction force microscopy. This integrated approach enabled the characterization of cell-generated traction stress and phenotypic expression in response to ECM cues. We investigated the impact of substrate stiffness and ECM composition on the differentiation of bipotential mouse embryonic liver (BMEL) progenitor cells. We observed that hepatocyte differentiation was primarily regulated by ECM composition, and cholangiocyte differentiation was cooperatively influenced by ECM proteins and stiffness properties. In particular, stiffness-mediated cholangiocyte differentiation was observed for cells cultured on fibronectin, while collagen IV promoted differentiation independent of substrate stiffness. We demonstrated the influence of cell contractility and traction stress in early cholangiocyte specification and further uncovered the roles of ERK and ROCK in this differentiation process. Overall, these findings illustrate the involvement of biomechanical signals in liver progenitor differentiation. Further, this approach could enable investigations for a broad range of cell types and ECM proteins, providing an integrated platform for evaluating the combinatorial effects of biochemical and biophysical signals in cell differentiation.

  11. To Broad-Match or Not to Broad-Match : An Auctioneer's Dilemma ?

    CERN Document Server

    Singh, Sudhir Kumar

    2008-01-01

    We initiate the study of an interesting aspect of sponsored search advertising, namely the consequences of broad match-a feature where an ad of an advertiser can be mapped to a broader range of relevant queries, and not necessarily to the particular keyword(s) that ad is associated with. Starting with a very natural setting for strategies available to the advertisers, and via a careful look through algorithmic and complexity theoretic glasses, we first propose a solution concept called broad match equilibrium(BME) for the game originating from the strategic behavior of advertisers as they try to optimize their budget allocation across various keywords. Next, we consider two broad match scenarios based on factors such as information asymmetry between advertisers and the auctioneer, and the extent of auctioneer's control on the budget splitting. In the first scenario, the advertisers have the full information about broad match and relevant parameters, and can reapportion their own budgets to utilize the extra i...

  12. Robust plasmonic substrates

    DEFF Research Database (Denmark)

    Kostiučenko, Oksana; Fiutowski, Jacek; Tamulevicius, Tomas;

    2014-01-01

    substrates is presented, which relies on the coverage of gold nanostructures with diamond-like carbon (DLC) thin films of thicknesses 25, 55 and 105 nm. DLC thin films were grown by direct hydrocarbon ion beam deposition. In order to find the optimum balance between optical and mechanical properties...... and breaking. DLC coating with thicknesses between 25 and 105 nm is found to considerably increase the mechanical strength of the substrates while at the same time ensuring conservation of sufficient field enhancements of the gold plasmonic substrates....

  13. Broad spectrum antibiotic compounds and use thereof

    Energy Technology Data Exchange (ETDEWEB)

    Koglin, Alexander; Strieker, Matthias

    2016-07-05

    The discovery of a non-ribosomal peptide synthetase (NRPS) gene cluster in the genome of Clostridium thermocellum that produces a secondary metabolite that is assembled outside of the host membrane is described. Also described is the identification of homologous NRPS gene clusters from several additional microorganisms. The secondary metabolites produced by the NRPS gene clusters exhibit broad spectrum antibiotic activity. Thus, antibiotic compounds produced by the NRPS gene clusters, and analogs thereof, their use for inhibiting bacterial growth, and methods of making the antibiotic compounds are described.

  14. Broad spectrum microarray for fingerprint-based bacterial species identification

    Directory of Open Access Journals (Sweden)

    Frey Jürg E

    2010-02-01

    Full Text Available Abstract Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups.

  15. Engineering broadly neutralizing antibodies for HIV prevention and therapy.

    Science.gov (United States)

    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options. PMID:26827912

  16. Broad band spectroscopic ellipsometry for the characterization of photovoltaic materials

    Energy Technology Data Exchange (ETDEWEB)

    Abou-Elfotouh, F.A.; Horner, G.S.; Coutts, T.J.; Wanlass, M.W. (Solar Energy Research Inst., Golden, CO (USA))

    1991-05-01

    The availability of commercial spectroscopic ellipsometers (SE) has been restricted to the UV-visible range from 250-900 nm. Although this is useful for many applications, it must be extended to the near IR region (up to 1700 nm) for the study of the optical behavior of most photovoltaic materials. This paper discusses the development of a broad band (300-1700 nm) SE which has been used to measure the optical characteristics of various materials. Among these are the polycrystalline thin film materials, CuInSe{sub 2} and CdTe (for which single crystal samples have also been investigated), and materials for high efficiency cascade solar cells including InP, InGaAs and InGaAsP. Most of these data are not presently available over such a wide spectral range. Experimentally, a rotating polarizer-fixed analyzer ellipsometer with an a.c. detection system has been developed for accurate measurement of psi and {Delta}, the relevant ellipsometric parameters, in the near IR. This approach has certain advantages over the rotating analyzer-fixed polarizer systems including reduced sensitivity to room light. The analytical methods include the use of a specially developed computer modeling program which gives psi and {Delta} for a given set of values related to the film thickness (which may be finite or zero) and to the optical properties of the substrate. (orig.).

  17. Against a Broad Definition of "Empathy"

    Directory of Open Access Journals (Sweden)

    Sarah Songhorian

    2015-04-01

    Full Text Available In this paper I will try to provide some arguments against a broad definition of “empathy”. Firstly, I will deal with attempts to define empathy as an umbrella concept. Then, I will try to point out the four main elements which contribute to the confusion that researchers in both the social and political as well as the scientific and philosophical domains face when dealing with empathy. In order to resolve this confusion, I suggest applying David Marr’s distinction to the field of empathy. Instead of providing an umbrella definition for empathy, which tries to account for all the data coming from different disciplines, I believe understanding that there are different levels of explanations and that different disciplines can contribute to each of them will provide a more detailed and less confused definition of empathy.

  18. Broad-band acoustic hyperbolic metamaterial

    CERN Document Server

    Shen, Chen; Sui, Ni; Wang, Wenqi; Cummer, Steven A; Jing, Yun

    2015-01-01

    Acoustic metamaterials (AMMs) are engineered materials, made from subwavelength structures, that exhibit useful or unusual constitutive properties. There has been intense research interest in AMMs since its first realization in 2000 by Liu et al. A number of functionalities and applications have been proposed and achieved using AMMs. Hyperbolic metamaterials are one of the most important types of metamaterials due to their extreme anisotropy and numerous possible applications, including negative refraction, backward waves, spatial filtering, and subwavelength imaging. Although the importance of acoustic hyperbolic metamaterials (AHMMs) as a tool for achieving full control of acoustic waves is substantial, the realization of a broad-band and truly hyperbolic AMM has not been reported so far. Here, we demonstrate the design and experimental characterization of a broadband AHMM that operates between 1.0 kHz and 2.5 kHz.

  19. A broad view of model validation

    International Nuclear Information System (INIS)

    The safety assessment of a nuclear waste repository requires the use of models. Such models need to be validated to ensure, as much as possible, that they are a good representation of the actual processes occurring in the real system. In this paper we attempt to take a broad view by reviewing step by step the modeling process and bringing out the need to validating every step of this process. This model validation includes not only comparison of modeling results with data from selected experiments, but also evaluation of procedures for the construction of conceptual models and calculational models as well as methodologies for studying data and parameter correlation. The need for advancing basic scientific knowledge in related fields, for multiple assessment groups, and for presenting our modeling efforts in open literature to public scrutiny is also emphasized. 16 refs

  20. Magnetohydrodynamic stability of broad line region clouds

    CERN Document Server

    Krause, Martin; Burkert, Andreas

    2012-01-01

    Hydrodynamic stability has been a longstanding issue for the cloud model of the broad line region in active galactic nuclei. We argue that the clouds may be gravitationally bound to the supermassive black hole. If true, stabilisation by thermal pressure alone becomes even more difficult. We further argue that if magnetic fields should be present in such clouds at a level that could affect the stability properties, they need to be strong enough to compete with the radiation pressure on the cloud. This would imply magnetic field values of a few Gauss for a sample of Active Galactic Nuclei we draw from the literature. We then investigate the effect of several magnetic configurations on cloud stability in axi-symmetric magnetohydrodynamic simulations. For a purely azimuthal magnetic field which provides the dominant pressure support, the cloud first gets compressed by the opposing radiative and gravitational forces. The pressure inside the cloud then increases, and it expands vertically. Kelvin-Helmholtz and colu...

  1. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate

    DEFF Research Database (Denmark)

    Grønborg, Mads; Kristiansen, Troels Zakarias; Stensballe, Allan;

    2002-01-01

    proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine...

  2. A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

    DEFF Research Database (Denmark)

    Weyer, Kathrin; Boldt, Henning Bünsow; Poulsen, Christine Bruun;

    2007-01-01

    Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP...

  3. Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV)

    DEFF Research Database (Denmark)

    Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall

    2011-01-01

    Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questio...

  4. PREFACE: Cell-substrate interactions Cell-substrate interactions

    Science.gov (United States)

    Gardel, Margaret; Schwarz, Ulrich

    2010-05-01

    not on the amount of ligand for adhesion receptors, but on its spatial distribution [1]. New protocols for the preparation of soft elastic substrates were essential to show that adhesion structures and cytoskeleton of adherent cells strongly adapt to substrate stiffness [2], with dramatic effects for cellular decision making. For example, it has been shown recently that differentiation of mesenchymal stem cells is strongly influenced by substrate stiffness [3]. Thus, physical factors appear to be equally important as biochemical ones in determining the cellular response to its substrate [4]. The introduction of novel physical techniques not only opened up completely new perspectives regarding biological function, it also introduced a new quantitative element into this field. For example, the availability of soft elastic substrates with controlled stiffness allows us to reconstruct cellular traction forces and to correlate them with other cellular features. This development enables modeling approaches to work in close contact with experimental data, thus opening up the perspective that the field of cell-substrate interactions will become a quantitative and predictive science in the future. Because physical research into cell-substrate interactions has become one of the fastest growing research areas in cellular biophysics and materials science, we believe that it is very timely that this special issue gathers some of the on-going research effort in this field. In contrast to the non-living world, cellular systems usually interact with their environment through specific adhesion, mainly based on adhesion receptors from the integrin family. During recent years, force spectroscopy has emerged as one of the main methods to study the physics of specific adhesion. In this special issue, single cell force spectroscopy is used by Boettiger and Wehrle-Haller to characterize the strength of cell-matrix adhesion and how it is modulated by the glycocalyx [5], while Chirasatitsin

  5. Biaxially textured composite substrates

    Energy Technology Data Exchange (ETDEWEB)

    Groves, James R.; Foltyn, Stephen R.; Arendt, Paul N.

    2005-04-26

    An article including a substrate, a layer of a metal phosphate material such as an aluminum phosphate material upon the surface of the substrate, and a layer of an oriented cubic oxide material having a rock-salt-like structure upon the metal phosphate material layer is provided together with additional layers such as a HTS top-layer of YBCO directly upon a layer of a buffer material such as a SrTi.sub.x Ru.sub.1-x O.sub.3 layer.

  6. Antibiofilm Peptides: Potential as Broad-Spectrum Agents.

    Science.gov (United States)

    Pletzer, Daniel; Hancock, Robert E W

    2016-10-01

    The treatment of bacterial diseases is facing twin threats, with increasing bacterial antibiotic resistance and relatively few novel compounds or strategies under development or entering the clinic. Bacteria frequently grow on surfaces as biofilm communities encased in a polymeric matrix. The biofilm mode of growth is associated with 65 to 80% of all clinical infections. It causes broad adaptive changes; biofilm bacteria are especially (10- to 1,000-fold) resistant to conventional antibiotics and to date no antimicrobials have been developed specifically to treat biofilms. Small synthetic peptides with broad-spectrum antibiofilm activity represent a novel approach to treat biofilm-related infections. Recent developments have provided evidence that these peptides can inhibit even developed biofilms, kill multiple bacterial species in biofilms (including the ESKAPE [Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species] pathogens), show strong synergy with several antibiotics, and act by targeting a universal stress response in bacteria. Thus, these peptides represent a promising alternative treatment to conventional antibiotics and work effectively in animal models of biofilm-associated infections. PMID:27068589

  7. Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

    OpenAIRE

    Kern, Melanie; Eisel, Florian; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2009-01-01

    Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Esche...

  8. Photoionisation modelling of the broad line region

    Science.gov (United States)

    King, Anthea

    2016-08-01

    Two of the most fundamental questions regarding the broad line region (BLR) are "what is its structure?" and "how is it moving?" Baldwin et al. (1995) showed that by summing over an ensemble of clouds at differing densities and distances from the ionising source we can easily and naturally produce a spectrum similar to what is observed for AGN. This approach is called the `locally optimally emitting clouds' (LOC) model. This approach can also explain the well-observed stratification of emission lines in the BLR (e.g. Clavel et al. 1991, Peterson et al. 1991, Kollatschny et al. 2001) and `breathing' of BLR with changes in the continuum luminosity (Netzer & Mor 1990, Peterson et al. 2014) and is therefore a generally accepted model of the BLR. However, LOC predictions require some assumptions to be made about the distribution of the clouds within the BLR. By comparing photoionization predictions, for a distribution of cloud properties, with observed spectra we can infer something about the structure of the BLR and distribution of clouds. I use existing reverberation mapping data to constrain the structure of the BLR by observing how individual line strengths and ratios of different lines change in high and low luminosity states. I will present my initial constraints and discuss the challenges associated with the method.

  9. Neutralization of low energy broad ion beam

    International Nuclear Information System (INIS)

    The paper is devoted to experimental and theoretical investigation of a low energy broad ion beam space charge and current compensation and ion-beam plasma (IBP), which would be created in transport space of the beam. The beam had cylindrical symmetry. The continuous uniform and hole tube like ion beams are used in the experiments. Different channels of electron appearing have been investigated for cases of neutralization due to secondary γ-electrons from the target and by electrons from glow cathode-neutralizer with metal or dielectric target. Results of neutralizing electrons energy distributions function measurements are presented as well as dependences of electron temperature and self-consisted plasma potential vs. beam parameters, ambient gas pressure, neutralizer parameters. Role of the thermoelectrons and dependence of IBP parameters on neutralizer area, location and potential are discussed. Significant role in neutralization of spatial collisional processes has been revealed even in neutralization by thermocathode. On the base of the experimental results self-consistent theoretical model have been developed, which describes the behavior of intense ion beam passing through the neutral gas at low pressure within conductive walls. The collisionless approach is used which means absence of collisional relaxation of the beam. This theory is used to derive the plasma potential and electron temperature within the beam

  10. Modeling of optical wireless scattering communication channels over broad spectra.

    Science.gov (United States)

    Liu, Weihao; Zou, Difan; Xu, Zhengyuan

    2015-03-01

    The air molecules and suspended aerosols help to build non-line-of-sight (NLOS) optical scattering communication links using carriers from near infrared to visible light and ultraviolet bands. This paper proposes channel models over such broad spectra. Wavelength dependent Rayleigh and Mie scattering and absorption coefficients of particles are analytically obtained first. They are applied to the ray tracing based Monte Carlo method, which models the photon scattering angle from the scatterer and propagation distance between two consecutive scatterers. Communication link path loss is studied under different operation conditions, including visibility, particle density, wavelength, and communication range. It is observed that optimum communication performances exist across the wavelength under specific atmospheric conditions. Infrared, visible light and ultraviolet bands show their respective features as conditions vary. PMID:26366662

  11. Ultra-Broad-Band Optical Parametric Amplifier or Oscillator

    Science.gov (United States)

    Strekalov, Dmitry; Matsko, Andrey; Savchenkov, Anatolly; Maleki, Lute

    2009-01-01

    A concept for an ultra-broad-band optical parametric amplifier or oscillator has emerged as a by-product of a theoretical study in fundamental quantum optics. The study was originally intended to address the question of whether the two-photon temporal correlation function of light [in particular, light produced by spontaneous parametric down conversion (SPDC)] can be considerably narrower than the inverse of the spectral width (bandwidth) of the light. The answer to the question was found to be negative. More specifically, on the basis of the universal integral relations between the quantum two-photon temporal correlation and the classical spectrum of light, it was found that the lower limit of two-photon correlation time is set approximately by the inverse of the bandwidth. The mathematical solution for the minimum two-photon correlation time also provides the minimum relative frequency dispersion of the down-converted light components; in turn, the minimum relative frequency dispersion translates to the maximum bandwidth, which is important for the design of an ultra-broad-band optical parametric oscillator or amplifier. In the study, results of an analysis of the general integral relations were applied in the case of an optically nonlinear, frequency-dispersive crystal in which SPDC produces collinear photons. Equations were found for the crystal orientation and pump wavelength, specific for each parametric-down-converting crystal, that eliminate the relative frequency dispersion of collinear degenerate (equal-frequency) signal and idler components up to the fourth order in the frequency-detuning parameter

  12. Broad spectrum anthelmintic potential of Cassia plants

    Institute of Scientific and Technical Information of China (English)

    Suman Kundu; Saptarshi Roy; Larisha Mawkhleing Lyndem

    2014-01-01

    Objective: To study the in vitro anthelmintic efficacy of Cassia alata (C. alata), Cassia(C. angustifolia) and Cassia occidentalis (C. occidentalis). angustifolia Methods: Crude ethanol extract from leaves of the three plants were prepared in rotary evaporator and different concentrations (10, 20 and 40 mg/mL) of leaf extracts were used for treatment on different representatives of helminthes (Heterakis gallinarum, Raillietina tetragona and Catatropis sp.) from domestic fowl (Gallus gallus domesticus). Loss of motility and death were monitored frequently.Results: C. alata showed early paralysis in all worms treated followed by C. angustifolia. C. occidentalis in combination with C. alata together caused early paralysis in all treated worms than the combination of C. alata with C. angustfolia. While Heterakis gallinarum in control survived for (81.33±2.07) h, treated worms lost their motility at (5.71±0.10) h, (6.60±0.86) h and (13.95±0.43) h with C. angustifolia, C. alata and C. occidentalis respectively at a concentration of 40 mg/mL which showed better efficacy than albendazole. Catatropis sp. survival period was (26.49±1.38) h in control, but with plant treatment, it lost its motility in just (0.57±0.08) h, (1.00±0.12) h and (1.47±0.40) h at 40 mg/mL concentration of C. alata, C. angustifolia and C. occidentalis respectively.Raillietina tetragona on the other hand became paralysed at (1.68±0.27) h, (2.95±0.29) h and (4.13±0.31) h with above concentrations treated with three plants respectively, however in control it survived up to (81.93±4.71) h.Conclusions:This present study indicated broad spectrum vermifugal activity of all plants tested.

  13. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  14. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

    Science.gov (United States)

    Nguyen, Giang K T; Wang, Shujing; Qiu, Yibo; Hemu, Xinya; Lian, Yilong; Tam, James P

    2014-09-01

    Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations. PMID:25038786

  15. Broad-Spectrum Solution-Processed Photovoltaics

    Science.gov (United States)

    Ip, Alexander Halley

    High global demand for energy coupled with dwindling fossil fuel supply has driven the development of sustainable energy sources such as solar photovoltaics. Emerging solar technologies aim for low-cost, solution-processable materials which would allow wide deployment. Colloidal quantum dots (CQDs) are such a materials system which exhibits the ability to absorb across the entire solar spectrum, including in the infrared where many technologies cannot harvest photons. However, due to their nanocrystalline nature, CQDs are susceptible to surface-associated electronic traps which greatly inhibit performance. In this thesis, surface engineering of CQDs is presented through a combined ligand approach which improves the passivation of surface trap states. A metal halide treatment is found to passivate quantum dot surfaces in solution, while bifunctional organic ligands produce a dense film in solid state. This approach reduced midgap trap states fivefold compared with conventional passivation strategies and led to solar cells with a record certified 7.0% power conversion efficiency. The effect of this process on the electronic structure is studied through photoelectron spectroscopy. It is found that while the halide provides deep trap passivation, the nature of the metal cation on the CQD surface affects the density of band tail states. This effect is explored further through a wide survey of materials, and it is found that the coordination ability of the metal cation is responsible for the suppression of shallow traps. With this understanding of CQD surface passivation, broad spectral usage is then explored through a study of visible-absorbing organolead halide perovskite materials as well as narrow-bandgap CQD solar cells. Control over growth conditions and modification of electrode interfaces resulted in efficient perovskite devices with effective usages of visible photons. For infrared-absorbing CQDs, it is found that, in addition to providing surface trap

  16. Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

    Science.gov (United States)

    Kern, Melanie; Eisel, Florian; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2012-01-01

    Summary Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active site HBMs (CX2CK or CX2CH). W. succinogenes CcsA2 was found to attach haem to standard CX2CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active site CX2CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX15CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterised structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome; a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes. PMID:19919672

  17. Transferring substrates to the 26S proteasome

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Seeger, Michael; Gordon, Colin

    2003-01-01

    Ubiquitin-dependent protein degradation is not only involved in the recycling of amino acids from damaged or misfolded proteins but also represents an essential and deftly controlled mechanism for modulating the levels of key regulatory proteins. Chains of ubiquitin conjugated to a substrate...... protein specifically target it for degradation by the 26S proteasome, a huge multi-subunit protein complex found in all eukaryotic cells. Recent reports have clarified some of the molecular mechanisms involved in the transfer of ubiquitinated substrates from the ubiquitination machinery to the proteasome...

  18. Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

    NARCIS (Netherlands)

    Biesebeke, R. te; Boussier, A.; Biezen, N. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2006-01-01

    Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media

  19. Substrate recognition by the 2-hydroxycarboxylate transport proteins

    NARCIS (Netherlands)

    Bandell, Michael

    2000-01-01

    The object of the research described in this thesis was to identify substrate-protein interactions that provide affinity and specificity for CitPLEME from Lc.mesenteroides and MlePLALA form L.lactis. The ability of these transporters to transport substrates that differ in structure and charge implie

  20. Printed electronic on flexible and glass substrates

    Science.gov (United States)

    Futera, Konrad; Jakubowska, Małgorzata; Kozioł, Grażyna

    2010-09-01

    Organic electronics is a platform technology that enables multiple applications based on organic electronics but varied in specifications. Organic electronics is based on the combination of new materials and cost-effective, large area production processes that provide new fields of application. Organic electronic by its size, weight, flexibility and environmental friendliness electronics enables low cost production of numerous electrical components and provides for such promising fields of application as: intelligent packaging, low cost RFID, flexible solar cells, disposable diagnostic devices or games, and printed batteries [1]. The paper presents results of inkjetted electronics elements on flexible and glass substrates. The investigations was target on characterizing shape, surface and geometry of printed structures. Variety of substrates were investigated, within some, low cost, non specialized substrate, design for other purposes than organic electronic.

  1. Nitrification in a zeoponic substrate

    Science.gov (United States)

    McGilloway, R. L.; Weaver, R. W.; Ming, D. W.; Gruener, J. E.

    2003-01-01

    Clinoptilolite is a zeolite mineral with high cation exchange capacity used in zeoponic substrates that have been proposed as a solid medium for growing plants or as a fertilizer material. The kinetics of nitrification has not been measured for NH4+ saturated zeoponic substrate. Experiments were conducted to evaluate the production of NO2- and NO3-, and nitrifier populations in zeoponic substrates. Small columns were filled with zeoponic substrate inoculated with a commercial inoculum or soil enrichment culture of nitrifying bacteria. In addition to column studies, a growth chamber study was conducted to evaluate the kinetics of nitrification in zeoponic substrates used to grow radishes (Raphanus sativus L.). The zeoponic substrate provided a readily available source of NH4+, and nitrifying bacteria were active in the substrate. Ammonium oxidation rates in column studies ranged from 5 to 10 micrograms N g-1 substrate h-1, and NO2- oxidation rates were 2 to 9.5 micrograms N g-1 substrate h-1. Rates determined from the growth chamber study were approximately 1.2 micrograms N g-1 substrate h-1. Quantities of NH4+ oxidized to NO2- and NO3- in inoculated zeoponic substrate were in excess of plant up-take. Acidification as a result of NH4+ oxidation resulted in a pH decline, and the zeoponic substrate showed limited buffering capacity.

  2. Multistructural biomimetic substrates for controlled cellular differentiation

    International Nuclear Information System (INIS)

    Multidimensional scaffolds are considered to be ideal candidates for regenerative medicine and tissue engineering based on their potential to provide an excellent microenvironment and direct the fate of the cultured cells. More recently, the use of stem cells in medicine has opened a new technological opportunity for controlled tissue formation. However, the mechanism through which the substrate directs the differentiation of stem cells is still rather unclear. Data concerning its specific surface chemistry, topology, and its signaling ability need to be further understood and analyzed. In our study, atomic force microscopy was used to study the stiffness, roughness, and topology of the collagen (Coll) and metallized collagen (MC) substrates, proposed as an excellent substrate for regenerative medicine. The importance of signaling molecules was studied by constructing a new hybrid signaling substrate that contains both collagen and laminin extracellular matrix (ECM) proteins. The cellular response—such as attachment capability, proliferation and cardiac and neuronal phenotype expression on the metallized and non-metallized hybrid substrates (collagen + laminin)—was studied using MTT viability assay and immunohistochemistry studies. Our findings indicate that such hybrid materials could play an important role in the regeneration of complex tissues. (paper)

  3. Maintainable substrate carrier for electroplating

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chen-An; Abas, Emmanuel Chua; Divino, Edmundo Anida; Ermita, Jake Randal G.; Capulong, Jose Francisco S.; Castillo, Arnold Villamor; Ma, Diana Xiaobing

    2016-08-02

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The carrier includes a non-conductive carrier body on which the substrates are placed and conductive lines embedded within the carrier body. A plurality of conductive clip attachment parts are attached in a permanent manner to the conductive lines embedded within the carrier body. A plurality of contact clips are attached in a removable manner to the clip attachment parts. The contact clips hold the substrates in place and conductively connecting the substrates with the conductive lines. Other embodiments, aspects and features are also disclosed.

  4. Tailored surfaces of perovskite oxide substrates for conducted growth of thin films.

    Science.gov (United States)

    Sánchez, Florencio; Ocal, Carmen; Fontcuberta, Josep

    2014-04-01

    Oxide electronics relies on the availability of epitaxial oxide thin films. The extreme flexibility of the chemical composition of ABO3 perovskites and the broad spectrum of properties they cover, inspire the creativity of scientists and place perovskites in the lead of functional materials for advanced technologies. Moreover, emerging properties are being discovered at interfaces between distinct perovskites that could not be anticipated on the basis of those of the adjacent epitaxial layers. All dreamed new prospects require the use of suitable substrates for epitaxial growth. Perovskite single crystals are the workhorses of this activity and understanding and controlling their surface properties have become critical. In this tutorial review we will chiefly focus on the impact of the morphology and composition of the surface of ABO3 perovskite substrates on the growth mechanisms and properties of thin films epitaxially grown on them. As SrTiO3 is the most popular substrate, we will mostly concentrate on describing the current understanding and achievements for it. Illustrative examples of other perovskite substrates (LaAlO3, LSAT and DyScO3) will be also included. We will show that distinct chemical terminations can exist on the surfaces used for growth and we will review methods employed either to select the most appropriate one for specific growth to allow, for instance, tailoring the ultimate outmost epilayer, or to induce self-ordering to engineer long-range nanoscale patterns of chemical terminations. We will demonstrate the capacity of this knowledge by the growth of low-dimensional organic and inorganic structures. PMID:24553667

  5. Experimental analysis of green roof substrate detention characteristics.

    Science.gov (United States)

    Yio, Marcus H N; Stovin, Virginia; Werdin, Jörg; Vesuviano, Gianni

    2013-01-01

    Green roofs may make an important contribution to urban stormwater management. Rainfall-runoff models are required to evaluate green roof responses to specific rainfall inputs. The roof's hydrological response is a function of its configuration, with the substrate - or growing media - providing both retention and detention of rainfall. The objective of the research described here is to quantify the detention effects due to green roof substrates, and to propose a suitable hydrological modelling approach. Laboratory results from experimental detention tests on green roof substrates are presented. It is shown that detention increases with substrate depth and as a result of increasing substrate organic content. Model structures based on reservoir routing are evaluated, and it is found that a one-parameter reservoir routing model coupled with a parameter that describes the delay to start of runoff best fits the observed data. Preliminary findings support the hypothesis that the reservoir routing parameter values can be defined from the substrate's physical characteristics.

  6. Experimental analysis of green roof substrate detention characteristics.

    Science.gov (United States)

    Yio, Marcus H N; Stovin, Virginia; Werdin, Jörg; Vesuviano, Gianni

    2013-01-01

    Green roofs may make an important contribution to urban stormwater management. Rainfall-runoff models are required to evaluate green roof responses to specific rainfall inputs. The roof's hydrological response is a function of its configuration, with the substrate - or growing media - providing both retention and detention of rainfall. The objective of the research described here is to quantify the detention effects due to green roof substrates, and to propose a suitable hydrological modelling approach. Laboratory results from experimental detention tests on green roof substrates are presented. It is shown that detention increases with substrate depth and as a result of increasing substrate organic content. Model structures based on reservoir routing are evaluated, and it is found that a one-parameter reservoir routing model coupled with a parameter that describes the delay to start of runoff best fits the observed data. Preliminary findings support the hypothesis that the reservoir routing parameter values can be defined from the substrate's physical characteristics. PMID:24135095

  7. Multifunctional epitaxial systems on silicon substrates

    Science.gov (United States)

    Singamaneni, Srinivasa Rao; Prater, John Thomas; Narayan, Jagdish

    2016-09-01

    Multifunctional heterostructures can exhibit a wide range of functional properties, including colossal magneto-resistance, magnetocaloric, and multiferroic behavior, and can display interesting physical phenomena including spin and charge ordering and strong spin-orbit coupling. However, putting this functionality to work remains a challenge. To date, most of the work reported in the literature has dealt with heterostructures deposited onto closely lattice matched insulating substrates such as DyScO3, SrTiO3 (STO), or STO buffered Si(100) using concepts of lattice matching epitaxy (LME). However, strain in heterostructures grown by LME is typically not fully relaxed and the layers contain detrimental defects such as threading dislocations that can significantly degrade the physical properties of the films and adversely affect the device characteristics. In addition, most of the substrates are incompatible with existing CMOS-based technology, where Si (100) substrates dominate. This review discusses recent advances in the integration of multifunctional oxide and non-oxide materials onto silicon substrates. An alternative thin film growth approach, called "domain matching epitaxy," is presented which identifies approaches for minimizing lattice strain and unwanted defects in large misfit systems (7%-25% and higher). This approach broadly allows for the integration of multifunctional materials onto silicon substrates, such that sensing, computation, and response functions can be combined to produce next generation "smart" devices. In general, pulsed laser deposition has been used to epitaxially grow these materials, although the concepts developed here can be extended to other deposition techniques, as well. It will be shown that TiN and yttria-stabilized zirconia template layers provide promising platforms for the integration of new functionality into silicon-based computer chips. This review paper reports on a number of thin-film heterostructure systems that span a

  8. Substrate specificity of hybrid modules from peptide synthetases

    NARCIS (Netherlands)

    Elsner, A; Engert, H; Saenger, W; Hamoen, L; Venema, G; Bernhard, F

    1997-01-01

    Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine activating module of surfactin synthetase were constructed by combining the t

  9. A Low Cost High Specific Stiffness Mirror Substrate Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The primary purpose of this proposal is to develop and demonstrate a new technology for manufacturing an ultra-low-cost precision optical telescope mirror which can...

  10. Photocatalytic water treatment: substrate-specific activity of titanium dioxide

    NARCIS (Netherlands)

    Sobral Romao, Joana Isabel

    2015-01-01

    In this thesis, heterogeneous photocatalysis is presented as a promising technology for water purification. Photocatalysis is revisited from an experimental and engineering point of view, comprising some theoretical fundamentals towards the commercialization of domestic appliances for water purifica

  11. Partially overlapping substrate specificities of staphylococcal group A sortases

    NARCIS (Netherlands)

    Sibbald, Mark J. J. B.; Yang, Xiao-Mei; Tsompanidou, Eleni; Qu, Di; Hecker, Michael; Becher, Dorte; Buist, Girbe; van Dijl, Jan Maarten

    2012-01-01

    Sortases catalyze the covalent attachment of proteins with a C-terminal LPxTG motif to the cell walls of Gram-positive bacteria. Here, we show that deletion of the srtA genes of Staphylococcus aureus and Staphylococcus epidermidis resulted in the dislocation of several LPxTG proteins from the cell w

  12. Convergence of broad-scale migration strategies in terrestrial birds.

    Science.gov (United States)

    La Sorte, Frank A; Fink, Daniel; Hochachka, Wesley M; Kelling, Steve

    2016-01-27

    Migration is a common strategy used by birds that breed in seasonal environments. Selection for greater migration efficiency is likely to be stronger for terrestrial species whose migration strategies require non-stop transoceanic crossings. If multiple species use the same transoceanic flyway, then we expect the migration strategies of these species to converge geographically towards the most optimal solution. We test this by examining population-level migration trajectories within the Western Hemisphere for 118 migratory species using occurrence information from eBird. Geographical convergence of migration strategies was evident within specific terrestrial regions where geomorphological features such as mountains or isthmuses constrained overland migration. Convergence was also evident for transoceanic migrants that crossed the Gulf of Mexico or Atlantic Ocean. Here, annual population-level movements were characterized by clockwise looped trajectories, which resulted in faster but more circuitous journeys in the spring and more direct journeys in the autumn. These findings suggest that the unique constraints and requirements associated with transoceanic migration have promoted the spatial convergence of migration strategies. The combination of seasonal atmospheric and environmental conditions that has facilitated the use of similar broad-scale migration strategies may be especially prone to disruption under climate and land-use change. PMID:26791618

  13. A Broad Set of Chromatin Factors Influences Splicing

    Science.gov (United States)

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  14. Assessing Eli Broad's Assault on Public School System Leadership

    Science.gov (United States)

    English, Fenwick W.; Crowder, Zan

    2012-01-01

    Eli Broad's approach to reforming urban public education does not recognize his own self-interest in promoting changes within such educational systems, a classic problem of misrecognition. The Broad agenda is an assault on the notion of the mission of public education as a service instead of a for-profit enterprise concerned with making money for…

  15. Social Cognition, Social Skill, and the Broad Autism Phenotype

    Science.gov (United States)

    Sasson, Noah J.; Nowlin, Rachel B.; Pinkham, Amy E.

    2013-01-01

    Social-cognitive deficits differentiate parents with the "broad autism phenotype" from non-broad autism phenotype parents more robustly than other neuropsychological features of autism, suggesting that this domain may be particularly informative for identifying genetic and brain processes associated with the phenotype. The current study…

  16. Boot Camp for Education CEOs: The Broad Foundation Superintendents Academy

    Science.gov (United States)

    Jehlen, Alain

    2012-01-01

    The Broad Foundation Superintendents Academy is the most prominent and most controversial training institute for school chiefs. The Academy is the flagship program of the Eli and Edythe Broad Foundation, the smallest of a triumvirate of corporate foundations that are at the heart of the billionaire campaign to remake public education in the image…

  17. Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

    NARCIS (Netherlands)

    Eerde, André van; Hamiaux, Cyril; Pérez, Javier; Parsot, Claude; Dijkstra, Bauke W.

    2004-01-01

    Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 f

  18. Validity of Context-Specific versus Broad Individual Differences in International Assignments

    Directory of Open Access Journals (Sweden)

    Julio E. Fernández Cueto

    2011-01-01

    Full Text Available La investigación previa sobre la selección de expatriados ha empleado factores de personalidad amplios para probar el valor de las variables de personalidad como predictors del desempeño en las asignaciones internacionales. En contraste, este studio se centra en el desarrollo y comprobación de la validez añadida de competencias específicas del contexto. A partir de la literatura sobre gestión de expatriados, una medida denominada Auto-evaluación de la Competencia Global fue creada, envolviendo competencias específicas del contexto que parecían importantes para las asignaciones internacionales. Usando regresión jerárquica, los resultados de 57 expatriados en la República Dominicana mostraron que estas competencias específicas del contexto predecían el desempeño más allá de los factores de personalidad amplios basados en los Big Five.

  19. Current models broadly neglect specific needs of biodiversity conservation in protected areas under climate change

    Directory of Open Access Journals (Sweden)

    Moloney Kirk A

    2011-05-01

    Full Text Available Abstract Background Protected areas are the most common and important instrument for the conservation of biological diversity and are called for under the United Nations' Convention on Biological Diversity. Growing human population densities, intensified land-use, invasive species and increasing habitat fragmentation threaten ecosystems worldwide and protected areas are often the only refuge for endangered species. Climate change is posing an additional threat that may also impact ecosystems currently under protection. Therefore, it is of crucial importance to include the potential impact of climate change when designing future nature conservation strategies and implementing protected area management. This approach would go beyond reactive crisis management and, by necessity, would include anticipatory risk assessments. One avenue for doing so is being provided by simulation models that take advantage of the increase in computing capacity and performance that has occurred over the last two decades. Here we review the literature to determine the state-of-the-art in modeling terrestrial protected areas under climate change, with the aim of evaluating and detecting trends and gaps in the current approaches being employed, as well as to provide a useful overview and guidelines for future research. Results Most studies apply statistical, bioclimatic envelope models and focus primarily on plant species as compared to other taxa. Very few studies utilize a mechanistic, process-based approach and none examine biotic interactions like predation and competition. Important factors like land-use, habitat fragmentation, invasion and dispersal are rarely incorporated, restricting the informative value of the resulting predictions considerably. Conclusion The general impression that emerges is that biodiversity conservation in protected areas could benefit from the application of modern modeling approaches to a greater extent than is currently reflected in the scientific literature. It is particularly true that existing models have been underutilized in testing different management options under climate change. Based on these findings we suggest a strategic framework for more effectively incorporating the impact of climate change in models exploring the effectiveness of protected areas.

  20. Validity of Context-Specific versus Broad Individual Differences in International Assignments

    OpenAIRE

    Julio E. Fernández Cueto; Juan I. Sánchez

    2011-01-01

    La investigación previa sobre la selección de expatriados ha empleado factores de personalidad amplios para probar el valor de las variables de personalidad como predictors del desempeño en las asignaciones internacionales. En contraste, este studio se centra en el desarrollo y comprobación de la validez añadida de competencias específicas del contexto. A partir de la literatura sobre gestión de expatriados, una medida denominada Auto-evaluación de la Competencia Global fue creada, envolviend...

  1. Broad Specificity Efflux pumps and Their Role in Multidrug Resistance of Gram Negative Bacteria

    OpenAIRE

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2011-01-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are producing a worldwide health problem. The continuous dissemination of «multi-drug resistant» (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria, the over-expression of efflux pumps that expel structurally-unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibio...

  2. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

    Science.gov (United States)

    The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

  3. Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses.

    Science.gov (United States)

    Cheng, Xiaofei; Li, Fangfang; Cai, Jianyu; Chen, Wei; Zhao, Nan; Sun, Yuqiang; Guo, Yushuang; Yang, Xiuling; Wu, Xiaoyun

    2015-08-01

    Transcription activator-like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses. PMID:26308041

  4. PLZT capacitor on glass substrate

    Energy Technology Data Exchange (ETDEWEB)

    Fairchild, Manuel Ray; Taylor, Ralph S.; Berlin, Carl W.; Wong, Celine Wk; Ma, Beihai; Balachandran, Uthamalingam

    2016-03-29

    A lead-lanthanum-zirconium-titanate (PLZT) capacitor on a substrate formed of glass. The first metallization layer is deposited on a top side of the substrate to form a first electrode. The dielectric layer of PLZT is deposited over the first metallization layer. The second metallization layer deposited over the dielectric layer to form a second electrode. The glass substrate is advantageous as glass is compatible with an annealing process used to form the capacitor.

  5. Compound-Specific Isotope Analysis of Nitroaromatic Contaminant Transformations by Nitroarene Dioxygenases

    Science.gov (United States)

    Pati, Sarah G.; Kohler, Hans-Peter E.; Hofstetter, Thomas B.

    2014-05-01

    Dioxygenation is an important biochemical reaction that often initiates the mineralization of recalcitrant organic contaminants such as nitroaromatic explosives, chlorinated benzenes, and polycyclic aromatic hydrocarbons. However, to assess the extent of dioxygenation in contaminated environments is difficult because of competing transformation processes and further reactions of the dioxygenation products. Compound-specific isotope analysis (CSIA) offers a new approach to reliably quantify biodegradation initiated by dioxygenation based on changes in stable isotope ratios of the pollutant. For CSIA it is essential to know the kinetic isotope effects (KIEs) pertinent to the dioxygenation mechanism of organic contaminants. Unfortunately, the range of KIEs of such reactions is poorly constrained although many dioxygenase enzymes with a broad substrate specificity have been reported. Dioxygenase enzymes usually exhibit complex reaction kinetics involving multiple substrates and substrate-specific binding modes which makes the determination of KIEs challenging. The goal of this study was to explore the magnitude and variability of 13C-, 2H-, and 15N-KIEs for the dioxygenation of one contaminant class, that is nitroaromatic contaminants (NACs). To this end, we investigated the C, H, and N isotope fractionation during the dioxygenation of nitrobenzene (NB), 2-nitrotoluene (2-NT), and 3-nitrotoluene (3-NT) by pure cultures, E. coli clones, cell extracts, and purified enzymes. From isotope fractionations measured in the substrates and reaction products, we determined dioxygenation KIEs for different combinations of the three substrates with nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO). The 13C-, 2H-, and 15N-KIEs for the dioxygenation of NB by NBDO were consistent for all experimental systems considered (i.e., Comamonas sp. Strain JS765, E. coli clones, cell extracts of E. coli clones, and purified NBDO). This observation suggests that the isotope

  6. A broad distribution of the alternative oxidase in microsporidian parasites.

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams

    2010-02-01

    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  7. Pedestal substrate for coated optics

    Science.gov (United States)

    Hale, Layton C.; Malsbury, Terry N.; Patterson, Steven R.

    2001-01-01

    A pedestal optical substrate that simultaneously provides high substrate dynamic stiffness, provides low surface figure sensitivity to mechanical mounting hardware inputs, and constrains surface figure changes caused by optical coatings to be primarily spherical in nature. The pedestal optical substrate includes a disk-like optic or substrate section having a top surface that is coated, a disk-like base section that provides location at which the substrate can be mounted, and a connecting cylindrical section between the base and optics or substrate sections. The connecting cylindrical section may be attached via three spaced legs or members. However, the pedestal optical substrate can be manufactured from a solid piece of material to form a monolith, thus avoiding joints between the sections, or the disk-like base can be formed separately and connected to the connecting section. By way of example, the pedestal optical substrate may be utilized in the fabrication of optics for an extreme ultraviolet (EUV) lithography imaging system, or in any optical system requiring coated optics and substrates with reduced sensitivity to mechanical mounts.

  8. SERS substrate and a method of providing a SERS substrate

    DEFF Research Database (Denmark)

    2011-01-01

    Source: US2011116089A A substrate primarily for SERS determination, the substrate has a number of elongate elements with a density of at least 1x108 elongate elements per cm2 and having metal coated tips. When the elements may be made to lean toward each other, such as by providing a drop...

  9. Friction and Shear Strength at the Nanowire–Substrate Interfaces

    Directory of Open Access Journals (Sweden)

    Gu Yi

    2009-01-01

    Full Text Available Abstract The friction and shear strength of nanowire (NW–substrate interfaces critically influences the electrical/mechanical performance and life time of NW-based nanodevices. Yet, very few reports on this subject are available in the literature because of the experimental challenges involved and, more specifically no studies have been reported to investigate the configuration of individual NW tip in contact with a substrate. In this letter, using a new experimental method, we report the friction measurement between a NW tip and a substrate for the first time. The measurement was based on NW buckling in situ inside a scanning electron microscope. The coefficients of friction between silver NW and gold substrate and between ZnO NW and gold substrate were found to be 0.09–0.12 and 0.10–0.15, respectively. The adhesion between a NW and the substrate modified the true contact area, which affected the interfacial shear strength. Continuum mechanics calculation found that interfacial shear strengths between silver NW and gold substrate and between ZnO NW and gold substrate were 134–139 MPa and 78.9–95.3 MPa, respectively. This method can be applied to measure friction parameters of other NW–substrate systems. Our results on interfacial friction and shear strength could have implication on the AFM three-point bending tests used for nanomechanical characterisation.

  10. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    Directory of Open Access Journals (Sweden)

    Dawn L Taylor

    Full Text Available Vibrio cholerae encodes six resistance-nodulation-division (RND efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

  11. Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

    Science.gov (United States)

    Taylor, Dawn L; Ante, Vanessa M; Bina, X Renee; Howard, Mondraya F; Bina, James E

    2015-01-01

    Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels. PMID:25695834

  12. Ultra-broad bandwidth parametric amplification at degeneracy.

    Science.gov (United States)

    Limpert, J; Aguergaray, C; Montant, S; Manek-Hönninger, I; Petit, S; Descamps, D; Cormier, E; Salin, F

    2005-09-19

    We report on a novel approach of ultra-broad bandwidth parametric amplification around degeneracy. A bandwidth of up to 400 nm centered around 800 nm is amplified in a BBO crystal by using chirped pump pulses with a bandwitdth as broad as 10 nm. A supercontinuum signal is generated in a microstructured fiber, having to first order a quadratic chirp, which is necessary to ensure temporal overlap of the interacting waves over this broad bandwidth. Furthermore, we discuss the potential of this approach for an octave-spanning parametric amplification.

  13. Broad Ligament Pregnancy - Success Story of a Laparoscopically Managed Case.

    Science.gov (United States)

    Nayar, Jayashree; Nair, Sobha S

    2016-07-01

    Abdominal pregnancies constitute 1% of ectopic pregnancies, among which broad ligament pregnancy is a rare form. The maternal mortality rate has been reported to be as high as 20%. The diagnosis is seldom established before surgery. Laparoscopic management of broad ligament ectopic pregnancy is the ideal form of treatment in appropriately selected patients. We present the case report of successful laparoscopic treatment of a 3x3.5cm broad ligament pregnancy. A search of literature shows that ours is the 6(th) case report of such a rare ectopic pregnancy managed endoscopically successfully. PMID:27630914

  14. Ultra-broad bandwidth parametric amplification at degeneracy.

    Science.gov (United States)

    Limpert, J; Aguergaray, C; Montant, S; Manek-Hönninger, I; Petit, S; Descamps, D; Cormier, E; Salin, F

    2005-09-19

    We report on a novel approach of ultra-broad bandwidth parametric amplification around degeneracy. A bandwidth of up to 400 nm centered around 800 nm is amplified in a BBO crystal by using chirped pump pulses with a bandwitdth as broad as 10 nm. A supercontinuum signal is generated in a microstructured fiber, having to first order a quadratic chirp, which is necessary to ensure temporal overlap of the interacting waves over this broad bandwidth. Furthermore, we discuss the potential of this approach for an octave-spanning parametric amplification. PMID:19498762

  15. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    Science.gov (United States)

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne

    2014-03-15

    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  16. Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

    Science.gov (United States)

    Linenberger, Kimberly J.; Bretz, Stacey Lowery

    2015-01-01

    Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

  17. A Novel Radiative Substrate Heater

    OpenAIRE

    CEYLAN, Abdullah; Kaynak, Erdal; ÖZCAN, Şadan; Firat, Tezer

    2004-01-01

    In this work, we have developed a new low cost radiative substrate heater for deposition of high-Tc superconducting thin films. It has all the features required for the preparation of high quality superconducting thin films. It is possible to reach ~ 800 °C substrate temperature with it by using only one 250 W halogen projector bulb.

  18. Soft substrates suppress droplet splashing

    CERN Document Server

    Howland, Christopher J; Style, Robert W; Castrejón-Pita, A A

    2015-01-01

    Droplets splash when they impact dry, flat substrates above a critical velocity that depends on parameters such as droplet size, viscosity and air pressure. We show that substrate stiffness also impacts the splashing threshold by imaging ethanol drops impacting silicone gels of different stiffnesses. Splashing is significantly suppressed: droplets on the softest substrates need over 70% more kinetic energy to splash than they do on rigid substrates. We show that splash suppression is likely to be due to energy losses caused by deformations of soft substrates during the first few microseconds of impact. We find that solids with Youngs modulus $\\lesssim O(10^5)$Pa suppress splashing, in agreement with simple scaling arguments. Thus materials like soft gels and elastomers can be used as simple coatings for effective splash prevention.

  19. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy.

    Science.gov (United States)

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing

    2015-01-01

    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy.

  20. Broad Spectrum Sanitizing Wipes with Food Additives Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Microcide proposes to develop novel multipurpose non-toxic sanitizing wipes that are aqueous based, have shelf life of 3-5 years, have broad spectrum microbicidal...

  1. AGN Broad Line Regions Scale with Bolometric Luminosity

    CERN Document Server

    Trippe, Sascha

    2015-01-01

    The masses of supermassive black holes in active galactic nuclei (AGN) can be derived spectroscopically via virial mass estimators based on selected broad optical/ultraviolet emission lines. These estimates commonly use the line width as a proxy for the gas speed and the monochromatic continuum luminosity as a proxy for the radius of the broad line region. However, if the size of the broad line region scales with bolometric rather than monochromatic AGN luminosity, mass estimates based on different emission lines will show a systematic discrepancy which is a function of the color of the AGN continuum. This has actually been observed in mass estimates based on H-alpha / H-beta and C IV lines, indicating that AGN broad line regions indeed scale with bolometric luminosity. Given that this effect seems to have been overlooked as yet, currently used single-epoch mass estimates are likely to be biased.

  2. Clues to Quasar Broad Line Region Geometry and Kinematics

    DEFF Research Database (Denmark)

    Vestergaard, Marianne; Wilkes, B. J.; Barthel, P. D.

    2000-01-01

    width to show significant inverse correlations with the fractional radio core-flux density, R, the radio axis inclination indicator. Highly inclined systems have broader line wings, consistent with a high-velocity field perpendicular to the radio axis. By contrast, the narrow line-core shows...... and with an accretion disk-wind emitting the broad lines. A spherical distribution of randomly orbiting broad-line clouds and a polar high-ionization outflow are ruled out....

  3. Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw

    DEFF Research Database (Denmark)

    Rosgaard, L.; Andric, Pavle; Dam-Johansen, Kim;

    2007-01-01

    glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM...... for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates......In this study, the applicability of a "fed-batch" strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low...

  4. Packaging and Performance of 980nm Broad Area Semiconductor Lasers

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    High power broad area semiconductor lasers have found increasing applications in pumping of solid state laser systems and fiber amplifiers, frequency doubling, medical systems and material processing.Packaging including the assembly design, process and thermal management, has a significant impact on the optical performance and reliability of a high power broad area laser. In this paper, we introduce the package structures and assembling process of 980nm broad area lasers and report the performances including output power, thermal behavior and far fields.We will report two types of high power broad area laser assemblies.One is a microchannel liquid cooled assembly and the other is a conduction cooled CT-mount assembly. Optical powers of 15W and 10W were achieved from a 980nm broad area laser with a 120 μ m stripe width in a microchannel liquid cooled assembly and conduction cooled CT-mount assembly, respectively.Furthermore,a high power of 6.5W out of fiber was demonstrated from a pigtailed, fully packaged butterfly-type module without TEC (Thermoelectric cooler).The measurement results showed that thermal management is the key in not only improving output power, but also significantly improving beam divergence and far field distribution.The results also showed that the die attach solder can significant impact the reliability of high power broad area lasers and that indium solder is not suitable for high power laser applications due to electromigration at high current densities and high temperatures.

  5. New imaging technologies to characterize arrhythmic substrate.

    Directory of Open Access Journals (Sweden)

    Antonio Dello Russo, MD, PhD; Sergio Conti, MD; Ghaliah Al-Mohani, MD, Phd; Michela Casella, MD, PhD; Francesca Pizzamiglio, MD; Corrado Carbucicchio, MD; Stefania Riva, MD; Gaetano Fassini, MD; Massimo Moltrasio, MD; Fabrizio Tundo, MD, PhD; Martina Zucchetti, MD; Benedetta Majocchi, MD; Eleonora Russo, MD; Vittoria Marino, MD; Maria Antonietta Dessanai MD; Fabrizio Bologna, MD; Luigi Di Biase, MD, PhD; Andrea Natale, MD; Claudio Tondo , MD, PhD.

    2014-12-01

    Full Text Available Abstract The cornerstone of the new imaging technologies to treat complex arrhythmias is the electroanatomic (EAM mapping. It is based on tissue characterization and in particular on determination of low potential region and dense scar definition. Recently, the identification of fractionated isolated late potentials increased the specificity of the information derived from EAM. In addition, non-invasive tools and their integration with EAM, such as cardiac magnetic resonance imaging and computed tomography scanning, have been shown to be helpful to characterize the arrhythmic substrate and to guide the mapping and the ablation. Finally, intracardiac echocardiography, known to be useful for several practical uses in the setting of electrophysiological procedures, it has been also demonstrated to provide important informations about the anatomical substrate and may have potential to identify areas of scarred myocardium.

  6. Optical substrate materials for synchrotron radiation beamlines

    International Nuclear Information System (INIS)

    The authors consider the materials choices available for making optical substrates for synchrotron radiation beam lines. They find that currently the optical surfaces can only be polished to the required finish in fused silica and other glasses, silicon, CVD silicon carbide, electroless nickel and 17-4 PH stainless steel. Substrates must therefore be made of one of these materials or of a metal that can be coated with electroless nickel. In the context of material choices for mirrors they explore the issues of dimensional stability, polishing, bending, cooling, and manufacturing strategy. They conclude that metals are best from an engineering and cost standpoint while the ceramics are best from a polishing standpoint. They then give discussions of specific materials as follows: silicon carbide, silicon, electroless nickel, Glidcop trademark, aluminum, precipitation-hardening stainless steel, mild steel, invar and superinvar. Finally they summarize conclusions and propose ideas for further research

  7. Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays

    OpenAIRE

    Lin, Baochuan; Wang, Zheng; Vora, Gary J.; Jennifer A. Thornton; Schnur, Joel M.; Thach, Dzung C.; Blaney, Kate M.; Ligler, Adam G.; Malanoski, Anthony P.; Santiago, Jose; Walter, Elizabeth A.; Agan, Brian K.; Metzgar, David; Seto, Donald; Daum, Luke T.

    2006-01-01

    The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation st...

  8. Substrate-Sensitive Graphene Oxidation.

    Science.gov (United States)

    Zhang, Zhuhua; Yin, Jun; Liu, Xiaofei; Li, Jidong; Zhang, Jiahuan; Guo, Wanlin

    2016-03-01

    The inertness of graphene toward reaction with ambient molecules is essential for realizing durable devices with stable performance. Many device applications require graphene to contact with substrates, but whose impact on the chemical property of graphene has been largely overlooked. Here, we combine comprehensive first-principles analyses with experiments to show that graphene oxidation is highly sensitive to substrates. Graphene remains inert on SiO2 and hexagonal boron nitride but becomes increasingly weak against oxidation on metal substrates because of enhanced charge transfer and chemical interaction between them. In particular, Ni and Co substrates lead to spontaneous oxidation of graphene, while a Cu substrate maximally promotes the oxygen diffusion on graphene, with an estimated diffusivity 13 orders of magnitude higher than that on freestanding graphene. Bilayer graphene is revealed to have high oxidation resistance independent of substrate and thus is a better choice for high-performance nanoelectronics. Our findings should be extendable to a wide spectrum of chemical functionalizations of two-dimensional materials mediated by substrates. PMID:26884318

  9. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

    Science.gov (United States)

    Hall, Mary P; Unch, James; Binkowski, Brock F; Valley, Michael P; Butler, Braeden L; Wood, Monika G; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B; Benink, Hélène A; Eggers, Christopher T; Slater, Michael R; Meisenheimer, Poncho L; Klaubert, Dieter H; Fan, Frank; Encell, Lance P; Wood, Keith V

    2012-11-16

    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes. PMID:22894855

  10. Substrate integrated antennas and arrays

    CERN Document Server

    Cheng, Yu Jian

    2015-01-01

    Substrate Integrated Antennas and Arrays provides a single source for cutting-edge information on substrate integrated circuits (SICs), substrate integrated waveguide (SIW) feeding networks, SIW slot array antennas, SIC traveling-wave antennas, SIW feeding antennas, SIW monopulse antennas, and SIW multibeam antennas. Inspired by the author's extensive research, this comprehensive book:Describes a revolutionary SIC-based antenna technique with the potential to replace existing antenna technologiesExamines theoretical and experimental results connected to electrical and mechanical performanceExp

  11. Direct cooled power electronics substrate

    Science.gov (United States)

    Wiles, Randy H [Powell, TN; Wereszczak, Andrew A [Oak Ridge, TN; Ayers, Curtis W [Kingston, TN; Lowe, Kirk T [Knoxville, TN

    2010-09-14

    The disclosure describes directly cooling a three-dimensional, direct metallization (DM) layer in a power electronics device. To enable sufficient cooling, coolant flow channels are formed within the ceramic substrate. The direct metallization layer (typically copper) may be bonded to the ceramic substrate, and semiconductor chips (such as IGBT and diodes) may be soldered or sintered onto the direct metallization layer to form a power electronics module. Multiple modules may be attached to cooling headers that provide in-flow and out-flow of coolant through the channels in the ceramic substrate. The modules and cooling header assembly are preferably sized to fit inside the core of a toroidal shaped capacitor.

  12. Substrate noise coupling in RFICs

    CERN Document Server

    Helmy, Ahmed

    2008-01-01

    Substrate Noise Coupling in RFICs addresses substrate noise coupling in RF and mixed signal ICs when used in a system on chip (SoC) containing digital ICs as well. This trend of integrating RF, mixed signal ICs with large digital ICs is found in many of today's commercial ICs such as single chip Wi-Fi or Bluetooth solutions and is expected to grow rapidly in the future. The book reports modeling and simulation techniques for substrate noise coupling effects in RFICs and introduces isolation structures and design guides to mitigate such effects with the ultimate goal of enhancing the yield of R

  13. Droplet dynamics on patterned substrates

    Indian Academy of Sciences (India)

    A Dupuis; J M Yeomans

    2005-06-01

    We present a lattice Boltzmann algorithm which can be used to explore the spreading of droplets on chemically and topologically patterned substrates. As an example we use the method to show that the final configuration of a drop on a substrate comprising hydrophobic and hydrophilic stripes can depend sensitively on the dynamical pathway by which the state is reached. We also consider a substrate covered with micron-scale posts and investigate how this can lead to superhydrophobic behaviour. Finally we model how a Namibian desert beetle collects water from the wind.

  14. Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution.

    Directory of Open Access Journals (Sweden)

    Seiya Watanabe

    Full Text Available Proline racemase (ProR is a member of the pyridoxal 5'-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE, which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372 from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR. This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241 and natural ProR (phenylalanine. The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline, and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

  15. Drying and substrate concentrations interact to inhibit decomposition of carbon substrates added to combusted Inceptisols from a boreal forest

    OpenAIRE

    German, DP; Allison, SD

    2015-01-01

    © 2015, Springer-Verlag Berlin Heidelberg. Climate change is expected to alter the mechanisms controlling soil organic matter (SOM) stabilization. Under climate change, soil warming and drying could affect the enzymatic mechanisms that control SOM turnover and dependence on substrate concentration. Here, we used a greenhouse climate manipulation in a mature boreal forest soil to test two specific hypotheses: (1) Rates of decomposition decline at lower substrate concentrations, and (2) reducti...

  16. Film Growth on Nanoporous Substrate

    Science.gov (United States)

    Zhang, Xue; Joy, James; Zhao, Chenwei; Xu, J. M.; Valles, James

    Self-ordered nanoporous anodic aluminum oxide (AAO) provides an easy way to fabricate nano structured material, such as nano wires and nano particles. We employ AAO as substrates and focus on the thermally evaporated film growth on the surface of the substrate. With various materials deposited onto the substrate, we find the films show different structures, e,g. ordered array of nano particles for Lead and nanohoneycomb structure for Silver. We relate the differing behaviors to the difference of surface energy and diffusion constant. To verify this, the effect of substrate temperature on the film growth has been explored and the structure of the film has been successfully changed through the process. We are grateful for the support of NSF Grants No. DMR-1307290.

  17. Immune history profoundly affects broadly protective B cell responses to influenza

    Science.gov (United States)

    Andrews, Sarah F.; Huang, Yunping; Kaur, Kaval; Popova, Lyubov I.; Ho, Irvin Y.; Pauli, Noel T.; Dunand, Carole J. Henry; Taylor, William M; Lim, Samuel; Huang, Min; Qu, Xinyan; Lee, Jane-Hwei; Salgado-Ferrer, Marlene; Krammer, Florian; Palese, Peter; Wrammert, Jens; Ahmed, Rafi; Wilson, Patrick C.

    2016-01-01

    Generating a broadly protective influenza vaccine is critical to global health. Understanding how immune memory influences influenza immunity is central to this goal. We undertook an in-depth study of the B cell response to the pandemic 2009 H1N1 vaccine over consecutive years. Analysis of monoclonal Abs generated from vaccine-induced plasmablasts demonstrated that individuals with low preexisting serological titers to the vaccinating strain generated a broadly reactive, HA stalk-biased, response. Higher preexisting serum antibody levels correlated with a strain-specific HA head-dominated response. We demonstrate that this HA head immunodominance encompasses poor accessibility of the HA stalk epitopes. Further, we show polyreactivity of HA stalk-reactive antibodies that could cause counterselection of these cells. Thus, preexisting memory against HA head epitopes predominate, inhibiting a broadly protective response against the HA stalk upon revaccination with similar strains. Consideration of influenza exposure history is critical for new vaccine strategies designed to elicit broadly neutralizing antibodies. PMID:26631631

  18. Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis.

    Science.gov (United States)

    Lairson, Luke L; Watts, Andrew G; Wakarchuk, Warren W; Withers, Stephen G

    2006-12-01

    Despite their unparalleled catalytic prowess and environmental compatibility, enzymes have yet to see widespread application in synthetic chemistry. This lack of application and the resulting underuse of their enormous potential stems not only from a wariness about aqueous biological catalysis on the part of the typical synthetic chemist but also from limitations on enzyme applicability that arise from the high degree of substrate specificity possessed by most enzymes. This latter perceived limitation is being successfully challenged through rational protein engineering and directed evolution efforts to alter substrate specificity. However, such programs require considerable effort to establish. Here we report an alternative strategy for expanding the substrate specificity, and therefore the synthetic utility, of a given enzyme through a process of "substrate engineering". The attachment of a readily removable functional group to an alternative glycosyltransferase substrate induces a productive binding mode, facilitating rational control of substrate specificity and regioselectivity using wild-type enzymes. PMID:17057723

  19. Bid Optimization in Broad-Match Ad auctions

    CERN Document Server

    Even-dar, Eyal; Mirrokni, Vahab; Muthukrishnan, S; Nadav, Uri

    2009-01-01

    Ad auctions in sponsored search support ``broad match'' that allows an advertiser to target a large number of queries while bidding only on a limited number. While giving more expressiveness to advertisers, this feature makes it challenging to optimize bids to maximize their returns: choosing to bid on a query as a broad match because it provides high profit results in one bidding for related queries which may yield low or even negative profits. We abstract and study the complexity of the {\\em bid optimization problem} which is to determine an advertiser's bids on a subset of keywords (possibly using broad match) so that her profit is maximized. In the query language model when the advertiser is allowed to bid on all queries as broad match, we present an linear programming (LP)-based polynomial-time algorithm that gets the optimal profit. In the model in which an advertiser can only bid on keywords, ie., a subset of keywords as an exact or broad match, we show that this problem is not approximable within any ...

  20. Multilayer nanoparticle arrays for broad spectrum absorption enhancement in thin film solar cells

    CERN Document Server

    Krishnan, Aravind; Krishna, Siva Rama; Khan, Mohammed Zafar Ali

    2013-01-01

    In this paper, we present a theoretical study on the absorption efficiency enhancement of a thin film amorphous Silicon (a-Si) photovoltaic cell over a broad spectrum of wavelengths using multiple nanoparticle arrays. The light absorption efficiency is enhanced in the lower wavelengths by a nanoparticle array on the surface and in the higher wavelengths by another nanoparticle array embedded in the active region. The efficiency at intermediate wavelengths is enhanced by the constructive interference of plasmon coupled light. We optimize this design by tuning the radius of particles in both arrays, the period of the array and the distance between the two arrays. The optimization results in 61.44% increase in total quantum efficiency for a 500 nm thick a-Si substrate.

  1. Microstrip antenna on tunable substrate

    Science.gov (United States)

    Jose, K. A.; Varadan, Vijay K.; Varadan, Vasundara V.; Mohanan, P.

    1995-05-01

    The tunable patch antenna configurations are becoming popular and attractive in many aspects. This was mainly due to the advent of ferrite thin film technology and tunable substrate materials. The integration of monolithic microwave circuits and antennas are becoming easy today. In the development of magnetic tuning of microstrip patch on ferrite substrate is presented by Rainville and Harackewiez. Radiation characteristics of such antennas are presented by Pozer. Band width and radiation characteristics of such tunable antennas are measured and compared. Usually the substrate losses are considered in the analysis and metallization losses are assumed to be ideal. The analysis of magnetic tunable radiator including metallization and ferrite substrate losses are presented. However, all such tuning and integration of circuits and antennas are mainly on ferrite substrate due to magnetic tuning. Recently, Varadan et al. established that the BaxSr1-xTiO3 series ferroelectric materials such as Barium Strontium Titanate (BST) are well suited for microwave phase shifter applications. It could be possible to change the dielectric constant of these materials more than 50% depending on the BST composition, by changing the applied bias voltage. Also, the porosity of BST can be controlled during processing to produce dielectric constants in the range of 15 to 1500, with some trade off in tunability. In this paper, we are presenting the possibility of designing a microstrip patch antenna on such tunable substrate. Such antennas are having the major advantage of electronic tunability and compact size.

  2. Broad ligament pregnancy a diagnostic dilemma: a case report

    Directory of Open Access Journals (Sweden)

    Amit Gupta

    2016-07-01

    Full Text Available Broad ligament pregnancy is one of the rarest forms of ectopic pregnancy with high risk of maternal mortality. Although ultrasonography is usually helpful in making the diagnosis but it is mostly established during laparotomy. 34 year old G2P1 with previous caesarean section reported at 8th month of pregnancy with inability to perceive foetal movements. Ultrasonography confirmed intrauterine fetal demise. Patient was taken for caesarean section after failed induction. Intraoperative diagnosis of broad ligament pregnancy was made and broad ligament along with fetus, sac, fallopian tube and ovary was excised. Post-operative period was uneventful. [Int J Reprod Contracept Obstet Gynecol 2016; 5(7.000: 2478-2480

  3. Folded cavity angled-grating broad-area lasers.

    Science.gov (United States)

    Zhao, Yunsong; Zhu, Lin

    2013-10-01

    The angled-grating broad-area laser is a promising candidate for high power, high brightness diode laser source. The key point in the design is the angled gratings which can simultaneously support the unique snake-like zigzag lasing mode and eliminate the direct Fabry-Perot (FP) feedback. Unlike a conventional laser waveguide mode, the phase front of the zigzag mode periodically changes along the propagation direction. By use of the mirror symmetry of the zigzag mode, we propose and demonstrate the folded cavity angled-grating broad-area lasers. One benefit of this design is to reduce the required wafer space compared to a regular angled-grating broad-area laser, especially in a long cavity laser for high power operation. Experimental results show that the folded cavity laser exhibits good beam quality in far field with a slightly larger threshold and smaller slope efficiency due to the additional interface loss.

  4. Analysis of fuel system technology for broad property fuels

    Science.gov (United States)

    Coffinberry, G. A.

    1984-01-01

    An analytical study was performed in order to assess relative performance and economic factors involved with alternative advanced fuel systems for future commercial aircraft operating with broad property fuels. Significant results, with emphasis on design practicality from the engine manufacturer' standpoint, are highlighted. Several advanced fuel systems were modeled to determine as accurately as possible the relative merits of each system from the standpoint of compatibility with broad property fuel. Freezing point, thermal stability, and lubricity were key property issues. A computer model was formulated to determine the investment incentive for each system. Results are given.

  5. A GaAs monolithic low-noise broad-band amplifier

    Science.gov (United States)

    Archer, J. A.; Weidlich, H. P.; Pettenpaul, E.; Petz, F. A.; Huber, J.

    1981-12-01

    This paper describes the design, fabrication, and performance of GaAs monolithic low-noise broad-band amplifiers intended for broadcast receiver antenna amplifier, IF amplifier, and instrumentation applications. The process technology includes the use of Czochralski-grown semiinsulating substrates, localized implantation of ohmic and FET channel regions, and silicon nitride for passivation and MIM capacitors. The amplifiers employ shunt feedback to obtain input matching and flat broad-band response. One amplifier provides a gain of 24 dB, bandwidth of 930 MHz, and noise figure of 5.0 dB. A second amplifier provides a gain of 17 dB, bandwidth of 1400 MHz, and noise figure of 5.6 dB. Input and output VSWR's are typically less than 2:1 and the third-order intercept points are 28 and 32 dB, respectively. Improved noise figure and intercept point can be achieved by the use of external RF chokes.

  6. Phosphorus sorption capacities and physicochemical properties of nine substrate materials for constructed wetland

    Energy Technology Data Exchange (ETDEWEB)

    Cui, L.H.; Zhu, X.Z.; Ma, M.; Ouyang, Y.; Dong, M.; Zhu, W.L.; Luo, S.M. [South China Agricultural University, Guangdong (China)

    2008-08-15

    Constructed wetland (CW) is a promising technique for removal of pollutants from wastewater and agricultural runoff. The performance of a CW to remove pollutants, however, hinges on the use of suitable substrate materials. This study examined the physicochemical properties and phosphorus (P) sorption capacities of nine different CW substrate materials using both batch experiments and the Freundlich as well as the Langmuir isotherm. The nine substrate materials used in this study were turf, topsoil, gravel, midsized sand (MSS), blast furnace slag (BFS), coal burn slag (CBS), blast furnace artificial slag (BFAS), coal burn artificial slag (CBAS), and midsized artificial sand (MSAS). Experimental data showed that sorption of P increased with initial solution P concentrations for all nine substrate materials. The maximum P sorption capacity of the substrate materials estimated by Langmuir isotherm was in the following order: turf (4243 mg/kg substrate) > BFAS (2116 mg/kg substrate) > BFS (1598 mg/kg substrate) > CBS (1449 mg/kg substrate) > top soil (1396 mg/kg substrate) > CBAS (1194 mg/kg substrate) > MSAS (519 mg/kg substrate) > gravel (494 mg/kg substrate) > MSS (403 mg/kg substrate). The specific gravity of eight substrate materials (except gravel) had very significant negative correlations with the P sorption, whereas the particle diameter of D-60 and uniformity coefficient (K-60) had positive correlations with the P sorption. The cation exchange capacity, organic matter, available ferrous, and exchangeable aluminum of the eight substrate materials also had very significant positive correlations with the P sorption, while the pH of the substrate materials showed a very significant negative correlation with the P sorption. Our study further suggests that turf and CBAS are the two relatively ideal substrate materials suitable for removal of P from a CW system.

  7. Recent advances in designing substrate-competitive protein kinase inhibitors.

    Science.gov (United States)

    Han, Ki-Cheol; Kim, So Yeon; Yang, Eun Gyeong

    2012-01-01

    Protein kinases play central roles in cellular signaling pathways and their abnormal phosphorylation activity is inseparably linked with various human diseases. Therefore, modulation of kinase activity using potent inhibitors is an attractive strategy for the treatment of human disease. While most protein kinase inhibitors in clinical development are mainly targeted to the highly conserved ATP-binding sites and thus likely promiscuously inhibit multiple kinases including kinases unrelated to diseases, protein substrate-competitive inhibitors are more selective and expected to be promising therapeutic agents. Most substrate-competitive inhibitors mimic peptides derived from substrate proteins, or from inhibitory domains within kinases or inhibitor proteins. In addition, bisubstrate inhibitors are generated by conjugating substrate-competitive peptide inhibitors to ATP-competitive inhibitors to improve affinity and selectivity. Although structural information on protein kinases provides invaluable guidance in designing substrate-competitive inhibitors, other strategies including bioinformatics, computational modeling, and high-throughput screening are often employed for developing specific substrate-competitive kinase inhibitors. This review focuses on recent advances in the design and discovery of substrate-competitive inhibitors of protein kinases.

  8. Low-background temperature sensors fabricated on parylene substrates

    International Nuclear Information System (INIS)

    Temperature sensors fabricated from ultra-low radioactivity materials have been developed for low-background experiments searching for neutrinoless double-beta decay and the interactions of WIMP dark matter. The sensors consist of electrical traces photolithographically-patterned onto substrates of vapor-deposited parylene. They are demonstrated to function as expected, to do so reliably and robustly, and to be highly radio-pure. This work is a proof-of-concept study of a technology that can be applied to broad class of electronic circuits used in low-background experiments

  9. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  10. Intrinsic performance-limiting instabilities in two-level class-B broad-area lasers

    Science.gov (United States)

    Pakhomov, A. V.; Molevich, N. E.; Krents, A. A.; Anchikov, D. A.

    2016-08-01

    The present paper is concerned with the analytical and numerical investigation of the transverse spatio-temporal instabilities in two-level broad-area lasers for the specific class-B case. We show that the two-level class-B broad-area laser tends to naturally operate in the filamentary state. This is revealed to be provided with two causes. First of them is related with the homogeneous output profile being intrinsically unstable due to the traveling-wave instability, independently from the boundary conditions. Secondly, high sensitivity to the boundaries of the pumping region was found for the commonly used top-hat-like profile leading to boundaries-induced filamentary dynamics. Spatio-temporal properties of both instability mechanisms are studied and their effects on the resulting laser dynamical behavior are analyzed.

  11. Characterization of a radiographic system with broad energy band X-ray source

    International Nuclear Information System (INIS)

    High energy X-ray beams with broad band energy spectra allow performing radiographic analysis on different materials and objects of relevant interest that cannot be investigated with conventional X-ray sources. The quality of a radiographic image strongly depends on the characteristics of radiation source as the size of the X-ray emitting area, or focal spot, and the energy spectrum of the radiation. In this work the characterization of a broad-band energy Bremsstrahlung source obtained from a linac providing a 5.5 MeV electron beam colliding with a tungsten target is presented. In order to measure the focal-spot size an ad hoc slit camera has been designed and built and a specific technique was used. Furthermore an analysis of the energy spectrum of the beam was performed using a method based on X-ray diffraction by a mosaic crystal.

  12. Broad spectral range synchronized flat-top arrayed waveguide grating

    NARCIS (Netherlands)

    Akca, B. Imran; Doerr, Christopher R.; Pollnau, Markus; Ridder, de René M.

    2012-01-01

    A broad-band Mach-Zehnder-interferometer-synchronized flat-top arrayed waveguide grating is presented with a 0.5-dB bandwidth of 12 nm over 90 nm of spectral range and a central excess loss value of -0.5 dB.

  13. Model Invariance across Genders of the Broad Autism Phenotype Questionnaire

    Science.gov (United States)

    Broderick, Neill; Wade, Jordan L.; Meyer, J. Patrick; Hull, Michael; Reeve, Ronald E.

    2015-01-01

    ASD is one of the most heritable neuropsychiatric disorders, though comprehensive genetic liability remains elusive. To facilitate genetic research, researchers employ the concept of the broad autism phenotype (BAP), a milder presentation of traits in undiagnosed relatives. Research suggests that the BAP Questionnaire (BAPQ) demonstrates…

  14. Children and trauma : a broad perspective on exposure and recovery

    NARCIS (Netherlands)

    Alisic, E.

    2011-01-01

    The purpose of this dissertation was to generate a broad overview of children’s exposure to and recovery from trauma in order to promote theory building and the design of prevention and intervention activities. First, a general population study was conducted in 1770 primary school children. They fil

  15. Document understanding for a broad class of documents

    NARCIS (Netherlands)

    Aiello, Marco; Monz, Christof; Todoran, Leon; Worring, Marcel

    2002-01-01

    We present a document analysis system able to assign logical labels and extract the reading order in a broad set of documents. All information sources, from geometric features and spatial relations to the textual features and content are employed in the analysis. To deal effectively with these infor

  16. Broad-band hard X-ray reflectors

    DEFF Research Database (Denmark)

    Joensen, K.D.; Gorenstein, P.; Hoghoj, P.;

    1997-01-01

    Interest in optics for hard X-ray broad-band application is growing. In this paper, we compare the hard X-ray (20-100 keV) reflectivity obtained with an energy-dispersive reflectometer, of a standard commercial gold thin-film with that of a 600 bilayer W/Si X-ray supermirror. The reflectivity of ...

  17. Silver Nanoparticles with Broad Multiband Linear Optical Absorption

    KAUST Repository

    Bakr, Osman M.

    2009-07-06

    A simple one-pot method produces silver nanoparticles coated with aryl thiols that show intense, broad nonplasmonic optical properties. The synthesis works with many aryl-thiol capping ligands, including water-soluble 4-mercaptobenzoic acid. The nanoparticles produced show linear absorption that is broader, stronger, and more structured than most conventional organic and inorganic dyes.

  18. PRINCIPAR An Efficient, Broad-coverage, Principle-based Parser

    CERN Document Server

    Lin, D

    1994-01-01

    We present an efficient, broad-coverage, principle-based parser for English. The parser has been implemented in C++ and runs on SUN Sparcstations with X-windows. It contains a lexicon with over 90,000 entries, constructed automatically by applying a set of extraction and conversion rules to entries from machine readable dictionaries.

  19. Plasma enhanced diamond deposition on steel and Si substrates

    Institute of Scientific and Technical Information of China (English)

    Y.S. Li; Y. Tang; W. Chen; Q. Yang; C. Xiao; A. Hirose

    2009-01-01

    Diamond growth on Fe-Cr-Al-Si steel and Si substrates was comparatively investigated in microwave plasma enhanced chemical vapor deposition (MPCVD) reactor with different deposition parameters. Adherent nanocrystalline diamond films were directly deposited on this steel substrate under a typical deposition condition, whereas microcrystalline diamond films were produced on Si wafer. With increasing CH4 concentration, reaction pressure, or the total gas flow rate, the quality of nanocrystalline diamond films formed on Fe-Cr-Al-Si substrates is gradually deteriorated in terms of density and adhesion. This impaired diamond quality on steels is primarily associated with a combined effect by the substrate composition and the specific process conditions that favor excessive nucleation of diamond.

  20. Porous substrates filled with nanomaterials

    Science.gov (United States)

    Worsley, Marcus A.; Baumann, Theodore F.; Satcher, Jr., Joe H.; Stadermann, Michael

    2014-08-19

    A composition comprising: at least one porous carbon monolith, such as a carbon aerogel, comprising internal pores, and at least one nanomaterial, such as carbon nanotubes, disposed uniformly throughout the internal pores. The nanomaterial can be disposed in the middle of the monolith. In addition, a method for making a monolithic solid with both high surface area and good bulk electrical conductivity is provided. A porous substrate having a thickness of 100 microns or more and comprising macropores throughout its thickness is prepared. At least one catalyst is deposited inside the porous substrate. Subsequently, chemical vapor deposition is used to uniformly deposit a nanomaterial in the macropores throughout the thickness of the porous substrate. Applications include electrical energy storage, such as batteries and capacitors, and hydrogen storage.

  1. Human seminal proteinase and prostate-specific antigen are the same protein

    Indian Academy of Sciences (India)

    Abdul Waheed; Md Imtaiyaz Hassan; Robert L Van Etten; Faizan Ahmad

    2008-06-01

    Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32–34 kDa with protease activities. Based on some physicochemical, enzymatic and immunological properties, it is concluded that these proteins are in fact identical. The protein exhibits properties similar to kallikrein-like serine protease, trypsin, chymotrypsin and thiol acid protease. Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein. The results of this study should be useful in purifying and assaying this protein. Based on published studies and the present results, the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.

  2. Protein kinase substrate identification on functional protein arrays

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    2008-02-01

    Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

  3. Optimization of Fermentation Conditions of P.variabile JN-A525 and Substrate Specificity of Urethanase%变幻青霉氨基甲酸乙酯降解酶产酶条件优化及酶的底物特异性

    Institute of Scientific and Technical Information of China (English)

    谷晓蕾; 田亚平

    2013-01-01

    在单因素优化基础上,运用正交试验设计理论,优化氨基甲酸乙酯降解酶产生菌P.variabile JN-A525的产酶条件.优化发酵培养基配方为:葡萄糖20 g/L,牛肉膏10 g/L,KH2PO45 g/L、NaCl 1 g/L、KCl 5 g/L;发酵条件为:初始pH 6.0、培养温度32℃、接种体积分数3%、250 mL摇瓶装量70 mL、摇床转速100 r/min,在此优化条件下酶活比优化前提高了3.47倍.对经超声破碎、(Na)2SO4盐析、Superdex G-75凝胶过滤层析步骤初步纯化的酶之底物特异性的研究表明,在模拟黄酒体系下,该酶对尿素和氨基甲酸乙酯有相对强的降解作用,对一些酯类和氨基酸没有降解作用.%The fermentation process for P.variabile JN-A525 was optimized by single factor experiment and orthogonal tests.The results showed that the medium was composed of glucose 20 g/L,beef extract 10 g/L,KH2PO4 5 g/L,NaCl 1 g/L and KCl 5 g/L.The optimum fermentation conditions was 32 ℃,inoculum volume 3%,the optimum amount in flask 70/250 mL and the rotation speed of 100 r/min.Under these optimal conditions,the highest urethanase activity is 3.47 fold more than that of previous cinditions.After sonication methods,sodium-sulfate salting-out and superdex G-75 gel filtration chromatography,the urethanase from Penicillium variabile JN-A525 is further determined substrate specificity.Under simulated conditions,the urethanase has a relatively strong catalytic activity to urea and EC.Meanwhile,urethanase cannot catalyze the decomposition of amino acids and esters.

  4. Semiconductor films on flexible iridium substrates

    Science.gov (United States)

    Goyal, Amit

    2005-03-29

    A laminate semiconductor article includes a flexible substrate, an optional biaxially textured oxide buffer system on the flexible substrate, a biaxially textured Ir-based buffer layer on the substrate or the buffer system, and an epitaxial layer of a semiconductor. Ir can serve as a substrate with an epitaxial layer of a semiconductor thereon.

  5. Phonon scattering in graphene over substrate steps

    DEFF Research Database (Denmark)

    Sevincli, Haldun; Brandbyge, Mads

    2014-01-01

    We calculate the effect on phonon transport of substrate-induced bends in graphene. We consider bending induced by an abrupt kink in the substrate, and provide results for different step-heights and substrate interaction strengths. We find that individual substrate steps reduce thermal conductance...

  6. Sensor Technologies on Flexible Substrates

    Science.gov (United States)

    Koehne, Jessica

    2016-01-01

    NASA Ames has developed sensor technologies on flexible substrates integrated into textiles for personalized environment monitoring and human performance evaluation. Current technologies include chemical sensing for gas leak and event monitoring and biological sensors for human health and performance monitoring. Targeted integration include next generation EVA suits and flexible habitats.

  7. Modeling graphene-substrate interactions

    NARCIS (Netherlands)

    Amlaki, Taher

    2016-01-01

    In this thesis I focussed on the interactions between graphene-like materials (grapheme and germanene) and various substrates. The attractive properties of graphene like a high carrier mobility, its single-atomic thickness and its theoretical magic have made graphene a very popular and promising can

  8. Substrate preferences in laying hens

    NARCIS (Netherlands)

    Jong, de I.C.; Reenen, van K.

    2005-01-01

    We investigated the substrate preference of laying hens with respect to dustbathing and foraging behaviour, in order to determine which resources should be provided in laying hen housing systems for the expression of these behaviours. The consumer demand approach was used to study the strength of pr

  9. Neurobiological Substrates of Tourette's Disorder

    NARCIS (Netherlands)

    Leckman, James F.; Bloch, Michael H.; Smith, Megan E.; Larabi, Daouia; Hampson, Michelle

    2010-01-01

    Objective: This article reviews the available scientific literature concerning the neurobiological substrates of Tourette's disorder (TD). Methods: The electronic databases of PubMed, ScienceDirect, and PsycINFO were searched for relevant studies using relevant search terms. Results: Neuropathologic

  10. Substrate Recognition of Histone H2B by DUBm

    Science.gov (United States)

    Henderson, Elizabeth; Berndsen, Christopher; Wolberger, Cynthia

    2011-03-01

    The SAGA complex is a transcriptional coactivator that regulates gene expression in eukaryotes via histone acetylation and deubiquitination, which are crucial for transcription. Our lab is investigating the SAGA-dependent deubiquitination of histone H2B. The deubiquitinating module (DUBm) of SAGA is comprised of a ubiquitin-specific protease, Ubp8, and three other proteins. It is known that Ubp8 cleaves ubiquitin from histone H2B, however, the specific way in which the enzyme binds to the substrate remains elusive. In order to unravel this mechanism, we attempted to determine the crystal structure of the substrate binding complex. We obtained this substrate by exploiting the techniques of intein chemistry to artificially ubiquitinate a histone H2B peptide, which we then co-crystallized with DUBm. Additionally, we synthesized Ub-K63R-linked chains and Ub-K48-linked chains and co-crystallized them with DUBm.

  11. Quasar Cartography: from Black Hole to Broad Line Region Scales

    CERN Document Server

    Chelouche, Doron

    2013-01-01

    A generalized approach to reverberation mapping (RM) is presented, which is applicable to broad- and narrow-band photometric data, as well as to spectroscopic observations. It is based on multivariate correlation analysis techniques and, in its present implementation, is able to identify reverberating signals across the accretion disk and the broad line region (BLR) of active galactic nuclei (AGN). Statistical tests are defined to assess the significance of time-delay measurements using this approach, and the limitations of the adopted formalism are discussed. It is shown how additional constraints on some of the parameters of the problem may be incorporated into the analysis thereby leading to improved results. When applied to a sample of 14 Seyfert 1 galaxies having good-quality high-cadence photometric data, accretion disk scales and BLR sizes are simultaneously determined, on a case-by-case basis, in most objects. The BLR scales deduced here are in good agreement with the findings of independent spectrosc...

  12. Experimental evaluation of combustor concepts for burning broad property fuels

    Science.gov (United States)

    Kasper, J. M.; Ekstedt, E. E.; Dodds, W. J.; Shayeson, M. W.

    1980-01-01

    A baseline CF6-50 combustor and three advanced combustor designs were evaluated to determine the effects of combustor design on operational characteristics using broad property fuels. Three fuels were used in each test: Jet A, a broad property 13% hydrogen fuel, and a 12% hydrogen fuel blend. Testing was performed in a sector rig at true cruise and simulated takeoff conditions for the CF6-50 engine cycle. The advanced combustors (all double annular, lean dome designs) generally exhibited lower metal temperatures, exhaust emissions, and carbon buildup than the baseline CF6-50 combustor. The sensitivities of emissions and metal temperatures to fuel hydrogen content were also generally lower for the advanced designs. The most promising advanced design used premixing tubes in the main stage. This design was chosen for additional testing in which fuel/air ratio, reference velocity, and fuel flow split were varied.

  13. Flow characteristics at trapezoidal broad-crested side weir

    Directory of Open Access Journals (Sweden)

    Říha Jaromír

    2015-06-01

    Full Text Available Broad-crested side weirs have been the subject of numerous hydraulic studies; however, the flow field at the weir crest and in front of the weir in the approach channel still has not been fully described. Also, the discharge coefficient of broad-crested side weirs, whether slightly inclined towards the stream or lateral, still has yet to be clearly determined. Experimental research was carried out to describe the flow characteristics at low Froude numbers in the approach flow channel for various combinations of in- and overflow discharges. Three side weir types with different oblique angles were studied. Their flow characteristics and discharge coefficients were analyzed and assessed based on the results obtained from extensive measurements performed on a hydraulic model. The empirical relation between the angle of side weir obliqueness, Froude numbers in the up- and downstream channels, and the coefficient of obliqueness was derived.

  14. Shareholder, stakeholder-owner or broad stakeholder maximization

    DEFF Research Database (Denmark)

    Mygind, Niels

    2004-01-01

    With reference to the discussion about shareholder versus stakeholder maximization it is argued that the normal type of maximization is in fact stakeholder-owner maxi-mization. This means maximization of the sum of the value of the shares and stake-holder benefits belonging to the dominating...... stakeholder-owner. Maximization of shareholder value is a special case of owner-maximization, and only under quite re-strictive assumptions shareholder maximization is larger or equal to stakeholder-owner maximization. Broad stakeholder maximization is the sum of the returns to all stake-holders also...... including the shareholders of a company. Although it may be the ultimate goal for Corporate Social Responsibility to achieve this kind of maximization, broad stakeholder maximization is quite difficult to give a precise definition. There is no one-dimensional measure to add different stakeholder benefits...

  15. Broad Consent For Research With Biological Samples: Workshop Conclusions

    Science.gov (United States)

    Grady, Christine; Eckstein, Lisa; Berkman, Ben; Brock, Dan; Cook-Deegan, Robert; Fullerton, Stephanie M.; Greely, Hank; Hansson, Mats G.; Hull, Sara; Kim, Scott; Lo, Bernie; Pentz, Rebecca; Rodriguez, Laura; Weil, Carol; Wilfond, Benjamin S.; Wendler, David

    2016-01-01

    Different types of consent are used to obtain human biospecimens for future research. This variation has resulted in confusion regarding what research is permitted, inadvertent constraints on future research, and research proceeding without consent. The NIH Clinical Center’s Department of Bioethics held a workshop to consider the ethical acceptability of addressing these concerns by using broad consent for future research on stored biospecimens. Multiple bioethics scholars, who have written on these issues, discussed the reasons for consent, the range of consent strategies, gaps in our understanding, and concluded with a proposal for broad initial consent coupled with oversight and, when feasible, ongoing provision of information to donors. The manuscript describes areas of agreement as well as areas that need more research and dialogue. Given recent proposed changes to the Common Rule, and new guidance regarding storing and sharing data and samples, this is an important and timely topic. PMID:26305750

  16. Spectral Decomposition of Broad-Line AGNs and Host Galaxies

    CERN Document Server

    Vanden Berk, Daniel E; Yip, C W; Schneider, D P; Connolly, A J; Burton, R E; Jester, S; Hall, P B; Szalay, A S; Brinkmann, J; Berk, Daniel E. Vanden; Shen, Jiajian; Yip, Ching-Wa; Schneider, Donald P.; Connolly, Andrew J.; Burton, Ross E.; Jester, Sebastian; Hall, Patrick B.; Szalay, Alex S.; Brinkmann, John

    2005-01-01

    Using an eigenspectrum decomposition technique, we separate the host galaxy from the broad line active galactic nucleus (AGN) in a set of 4666 spectra from the Sloan Digital Sky Survey (SDSS), from redshifts near zero up to about 0.75. The decomposition technique uses separate sets of galaxy and quasar eigenspectra to efficiently and reliably separate the AGN and host spectroscopic components. The technique accurately reproduces the host galaxy spectrum, its contributing fraction, and its classification. We show how the accuracy of the decomposition depends upon S/N, host galaxy fraction, and the galaxy class. Based on the eigencoefficients, the sample of SDSS broad-line AGN host galaxies spans a wide range of spectral types, but the distribution differs significantly from inactive galaxies. In particular, post-starburst activity appears to be much more common among AGN host galaxies. The luminosities of the hosts are much higher than expected for normal early-type galaxies, and their colors become increasing...

  17. Oxygen consumption of rats with broad intestinal resection

    OpenAIRE

    Luz J.; Griggio M.A.; Fagundes D.J.; Araújo R.M.; Marcondes W.

    2000-01-01

    The study was performed to investigate possible alterations in oxygen consumption in an animal model with broad intestinal resection. Oxygen consumption and the thermal effect of a short meal were measured in rats subjected to short bowel syndrome. Four groups of rats were used. Group I was the control group, group II was sham operated, group III was submitted to 80% jejunum-ileum resection, and group IV was submitted to 80% jejunum-ileum resection with colon interposition. Ninety days after ...

  18. Ecological restoration of southwestern ponderosa pine ecosystems: A broad perspective

    OpenAIRE

    Allen, C. D.; Savage, M.; Falk, D. A.; Suckling, K. F.; Swetnam, T.W.; Schulke, T.; Stacey, P. B.; Morgan, P.; Hoffman, M; Klingel, J. T.

    2002-01-01

    The purpose of this paper is to promote a broad and flexible perspective on ecological restoration of Southwestern (U.S.) ponderosa pine forests. Ponderosa pine forests in the region have been radically altered by Euro-American land uses, including livestock grazing, fire suppression, and logging. Dense thickets of young trees now abound, old-growth and biodiversity have declined, and human and ecological communities are increasingly vulnerable to destructive crown fires. A consensus has emer...

  19. Flow structure in front of the broad-crested weir

    Directory of Open Access Journals (Sweden)

    Zachoval Zbyněk

    2015-01-01

    Full Text Available The paper deals with research focused on description of flow structure in front of broad-crested weir. Based on experimental measurement, the flow structure in front of the weir (the recirculation zone of flow and tornado vortices and flow structure on the weir crest has been described. The determined flow character has been simulated using numerical model and based on comparing results the suitable model of turbulence has been recommended.

  20. Series of broad resonances in atomic three-body systems

    CERN Document Server

    Diaz, D; Hu, C -Y

    2016-01-01

    We re-examine the series of resonances found earlier in atomic three-body systems by solving the Faddeev-Merkuriev integral equations. These resonances are rather broad and line-up at each threshold with gradually increasing gaps, the same way for all thresholds and irrespective of the spatial symmetry. We relate these resonances to the Gailitis mechanism, which is a consequence of the polarization potential.