Science.gov

Sample records for acetyltransferase reporter construct

  1. A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast.

    PubMed

    Mannhaupt, G; Pilz, U; Feldmann, H

    1988-07-30

    Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay. To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used. We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants. To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region. We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low. Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast.

  2. Construction quality assurance report

    SciTech Connect

    Roscha, V.

    1994-09-08

    This report provides a summary of the construction quality assurance (CQA) observation and test results, including: The results of the geosynthetic and soil materials conformance testing. The observation and testing results associates with the installation of the soil liners. The observation and testing results associated with the installation of the HDPE geomembrane liner systems. The observation and testing results associated with the installation of the leachate collection and removal systems. The observation and testing results associated with the installation of the working surfaces. The observation and testing results associated with in-plant manufacturing process. Summary of submittal reviews by Golder Construction Services, Inc. The submittal and certification of the piping material specifications. The observation and verification associated of the Acceptance Test Procedure results of the operational equipment functions. Summary of the ECNs which are incorporated into the project.

  3. Construction Report, 2000.

    ERIC Educational Resources Information Center

    Abramson, Paul

    2000-01-01

    Provides cost data on college and university construction projects completed for 1999 and projected for completion in 2000 by region. New building cost comparisons and profiles are listed, including basic information on 61 dormitory projects schedules to be completed in 2000. (GR)

  4. 17th Annual School Construction Report, 2012

    ERIC Educational Resources Information Center

    Abramson, Paul

    2012-01-01

    The 2012 "School Planning & Management"'s 17th Annual School Construction Report reports over the last two years although school construction had fallen from previous highs, the pipeline of projects funded before the recession was still full. And so, in 2009 total construction was a solid $16.4 billion. But the pipeline is not being…

  5. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    PubMed

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  6. Frugal Construction Standards. Final [Report].

    ERIC Educational Resources Information Center

    SMART Schools Clearinghouse, Tallahassee, FL.

    This booklet provides best practice recommendations for building functional and frugal schools in Florida. Seventeen best practice construction recommendations are addressed, including recommendations for sitework, concrete, masonry, metals, wood and plastics, thermal and moisture protection, doors and windows, finishes, equipment, furnishings,…

  7. 35th Annual Official Education Construction Report

    ERIC Educational Resources Information Center

    Agron, Joe

    2009-01-01

    Spending on construction by the nation's education institutions increased in 2008, reversing four years of declines in total annual expenditures, according to "American School & University"'s 35th annual Official Education Construction Report. One of the reasons for the increase in spending was the fact that the majority of the construction…

  8. 34th Annual Official Education Construction Report

    ERIC Educational Resources Information Center

    Agron, Joe

    2008-01-01

    According to American School & University's 34th annual Official Education Construction Report, spending on construction by the nation's school districts and colleges slowed in 2007, representing the fourth consecutive year total expenditures dropped from the year before and setting a low point in spending so far this decade. A difficult economy…

  9. 20th Annual Residence Hall Construction Report

    ERIC Educational Resources Information Center

    Agron, Joe

    2009-01-01

    Even in difficult economic times, colleges and universities continue to invest in residence hall construction projects as a way to attract new students and keep existing ones on campus. According to data from "American School & University"'s 20th annual Residence Hall Construction Report, the median new project completed in 2008 was…

  10. The lac operon galactoside acetyltransferase.

    PubMed

    Roderick, Steven L

    2005-06-01

    Of the proteins encoded by the three structural genes of the lac operon, the galactoside acetyltransferase (thiogalactoside transacetylase, LacA, GAT) encoded by lacA is the only protein whose biological role remains in doubt. Here, we briefly note the classical literature that led to the identification and initial characterization of GAT, and focus on more recent results which have revealed its chemical mechanism of action and its membership in a large superfamily of structurally similar acyltransferases. The structural and sequence similarities of several members of this superfamily confirm the original claim for GAT as a CoA-dependent acetyltransferase specific for the 6-hydroxyl group of certain pyranosides, but do not yet point to the identity of the natural substrate(s) of the enzyme.

  11. Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe ...

  12. Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

    SciTech Connect

    Brent, Michael M.; Iwata, Ayaka; Carten, Juliana; Zhao, Kehao; Marmorstein, Ronen

    2009-09-02

    The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys{sup 16} to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a 'primitive' model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-{angstrom} crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional 'bent helix' proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

  13. 16th Annual School Construction Report, 2011

    ERIC Educational Resources Information Center

    Abramson, Paul

    2011-01-01

    Almost 73 percent of construction in 2009 was of new school buildings. New school buildings accounted for less than 60 percent of construction spending in 2010, suggesting a shift to using less abundant construction dollars to upgrade and add to existing buildings. While overall construction fell more than $1.8 billion in 2010, spending on…

  14. 12th Annual School Construction Report, 2007

    ERIC Educational Resources Information Center

    Abramson, Paul

    2007-01-01

    School construction completed in 2006 totaled just more than $20 billion, a drop of seven percent from the record $21.6 billion put in place in 2005. Even so, it was the sixth year in the last seven that annual construction exceeded $20 billion. During the seven years of the present century, school districts have completed construction projects…

  15. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    DOEpatents

    Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  16. 1961-1968 New Construction Report.

    ERIC Educational Resources Information Center

    National Association of Physical Plant Administrators of Universities and Colleges, Richmond, IN.

    137 NAPPA colleges and universities provided data for this summary. Projects are summarized by thirteen building classifications. Under each classification the following information headings are used--(1) name of institution, (2) project completion date, (3) gross square feet, (4) net assignable area, (5) construction costs, (6) number of stories,…

  17. 19th Annual Residence Hall Construction Report

    ERIC Educational Resources Information Center

    Agron, Joe

    2008-01-01

    The construction of residence hall facilities at colleges and universities continues to be strong, as institutions scramble to meet the housing needs and varied demands of a growing student population. This article presents data collected from 39 new residence hall projects completed in 2007. According to American School & University's 19th…

  18. 18th Annual School Construction Report, 2013

    ERIC Educational Resources Information Center

    Abramson, Paul

    2013-01-01

    The bottom line on school construction in 2012 is that total spending edged up slightly from the previous year, (to $12.9 billion from $12.2 billion), but the spending for new schools declined from $6.9 billion to $6.177 billion. The increase in overall spending was attributable to more spending for additions and a major increase in spending for…

  19. Constructing a bit string universe. Progress report

    SciTech Connect

    Noyes, H.P.; Manthey, M.J.; Gefwert, C.

    1984-02-01

    We present a case for discrete, constructive physics that generates four scale constants, a connection to laboratory events and scattering theory that ties these to the dimensional constants c, h, m/sub p/, and G, and a tentative quantum number assignment consistent with standard model for leptons and quarks with three generations. Current first approximations from the theory are dirac constant c/e/sup 2/ = 137 +- O(1/137), dirac constant c/Gm/sup 2//sub p/ - 2/sup 127/ + 136 approx. = 1.7 x 10/sup 38/(1 +- 0(1/137)), and m/sub p//m/sub e/ = 1836.151497... +-. Our understanding of wave-particle dualism and observational cosmology creates no more experimental paradoxes than currently accepted views - perhaps fewer. 19 references.

  20. Enhanced Geothermal Systems (EGS) Well Construction Technology Evaluation Report

    SciTech Connect

    Polsky, Yarom; Capuano, Louis; Finger, John; Huh, Michael; Knudsen, Steve; Chip, A.J. Mansure; Raymond, David; Swanson, Robert

    2008-12-01

    This report provides an assessment of well construction technology for EGS with two primary objectives: 1. Determining the ability of existing technologies to develop EGS wells. 2. Identifying critical well construction research lines and development technologies that are likely to enhance prospects for EGS viability and improve overall economics.

  1. ORNL engineering design and construction reengineering report

    SciTech Connect

    McNeese, L.E.

    1998-01-01

    A team composed of individuals representing research and development (R and D) divisions, infrastructure support organizations, and Department of Energy (DOE)-Oak Ridge Operations was chartered to reengineer the engineering, design, and construction (ED and C) process at Oak Ridge National Laboratory (ORNL). The team recognized that ED and C needs of both R and D customers and the ORNL infrastructure program have to be met to maintain a viable and competitive national laboratory. Their goal was to identify and recommend implementable best-in-class ED and C processes that will efficiently and cost-effectively support the ORNL R and D staff by being responsive to their programmatic and infrastructure needs. The team conducted process mapping of current and potential ED and C approaches, developed idealized versions of ED and C processes, and identified potential barriers to an efficient ED and C process. Eight subteams were assigned to gather information and to evaluate the significance of potential barriers through benchmarking, surveys, interviews, and reviews of key topical areas in order to determine whether the perceived barriers were real and important and whether they resulted from laws or regulations over which ORNL has no control.

  2. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

    PubMed Central

    Watanabe, M; Igarashi, T; Kaminuma, T; Sofuni, T; Nohmi, T

    1994-01-01

    Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms. PMID:7889864

  3. Regulation and function of histone acetyltransferase MOF.

    PubMed

    Yang, Yang; Han, Xiaofei; Guan, Jingyun; Li, Xiangzhi

    2014-03-01

    The mammalian MOF (male absent on the first), a member of the MYST (MOZ, YBF2, SAS2, and Tip60) family of histone acetyltransferases (HATs), is the major enzyme that catalyzes the acetylation of histone H4 on lysine 16. Acetylation of K16 is a prevalent mark associated with chromatin decondensation. MOF has recently been shown to play an essential role in maintaining normal cell functions. In this study, we discuss the important roles of MOF in DNA damage repair, apoptosis, and tumorigenesis. We also analyze the role of MOF as a key regulator of the core transcriptional network of embryonic stem cells.

  4. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  5. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

    PubMed Central

    Dobransky, T; Davis, W L; Xiao, G H; Rylett, R J

    2000-01-01

    Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity. PMID:10861222

  6. DEMONSTRATION OF RADON RESISTANT CONSTRUCTION TECHNIQUES - PHASE II. FINAL REPORT

    EPA Science Inventory

    The report gives results of a demonstration of radon resistant construction techniques. Sub-slab mitigation systems were installed (in accordance with draft standards) in 15 new Florida houses in 1992, and these houses have undergone extensive testing to validate techniques used ...

  7. Live Virtual Constructive Distributed Test Environment Characterization Report

    NASA Technical Reports Server (NTRS)

    Murphy, Jim; Kim, Sam K.

    2013-01-01

    This report documents message latencies observed over various Live, Virtual, Constructive, (LVC) simulation environment configurations designed to emulate possible system architectures for the Unmanned Aircraft Systems (UAS) Integration in the National Airspace System (NAS) Project integrated tests. For each configuration, four scenarios with progressively increasing air traffic loads were used to determine system throughput and bandwidth impacts on message latency.

  8. A Larger Scale. Tenth Annual Residence Hall Construction Report.

    ERIC Educational Resources Information Center

    Argon, Joe

    1999-01-01

    Presents data from the American School & University's 10th Annual Residence Hall Construction Report that show dormitories are costing more per square foot to build while also becoming larger accommodations. Data tables are provided as are highlighted discussions that include residence hall design flexibility, environmental concerns and building…

  9. Body Awareness: Construct and Self-Report Measures

    PubMed Central

    Mehling, Wolf E.; Gopisetty, Viranjini; Daubenmier, Jennifer; Price, Cynthia J.; Hecht, Frederick M.; Stewart, Anita

    2009-01-01

    Objectives Heightened body awareness can be adaptive and maladaptive. Improving body awareness has been suggested as an approach for treating patients with conditions such as chronic pain, obesity and post-traumatic stress disorder. We assessed the psychometric quality of selected self-report measures and examined their items for underlying definitions of the construct. Data sources PubMed, PsychINFO, HaPI, Embase, Digital Dissertations Database. Review methods Abstracts were screened; potentially relevant instruments were obtained and systematically reviewed. Instruments were excluded if they exclusively measured anxiety, covered emotions without related physical sensations, used observer ratings only, or were unobtainable. We restricted our study to the proprioceptive and interoceptive channels of body awareness. The psychometric properties of each scale were rated using a structured evaluation according to the method of McDowell. Following a working definition of the multi-dimensional construct, an inter-disciplinary team systematically examined the items of existing body awareness instruments, identified the dimensions queried and used an iterative qualitative process to refine the dimensions of the construct. Results From 1,825 abstracts, 39 instruments were screened. 12 were included for psychometric evaluation. Only two were rated as high standard for reliability, four for validity. Four domains of body awareness with 11 sub-domains emerged. Neither a single nor a compilation of several instruments covered all dimensions. Key domains that might potentially differentiate adaptive and maladaptive aspects of body awareness were missing in the reviewed instruments. Conclusion Existing self-report instruments do not address important domains of the construct of body awareness, are unable to discern between adaptive and maladaptive aspects of body awareness, or exhibit other psychometric limitations. Restricting the construct to its proprio- and interoceptive

  10. Modular Design/Phased Construction Alternative Evaluation Report

    SciTech Connect

    Schwartztrauber, K.

    1999-05-28

    Modular design concepts are being considered for the license application during the surface facility design phase of the Monitored Geologic Repository (MGR). The Viability Assessment (VA) design is used as the reference design for the report. The primary objectives are to spread construction of the WHB and the subsurface repository over time to reduce annual project costs, and to provide a cost-effective design for the surface facilities that supports waste emplacement starting in the year 2010.

  11. The Novel SLIK Histone Acetyltransferase Complex Functions in the Yeast Retrograde Response Pathway

    PubMed Central

    Pray-Grant, Marilyn G.; Schieltz, David; McMahon, Stacey J.; Wood, Jennifer M.; Kennedy, Erin L.; Cook, Richard G.; Workman, Jerry L.; Yates III, John R.; Grant, Patrick A.

    2002-01-01

    The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAFIIs), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II. PMID:12446794

  12. N-Acetyltransferase 1 Polymorphism and Breast Cancer Risk

    DTIC Science & Technology

    2011-10-01

    analysis of the N-acetyltransferase 1 gene (NAT1*) using polymerase chain reaction-restriction fragment- single strand conformation polymorphism assay...risk of smoking-induced lung cancer (Bouchardy et al., 1998). NAT1*14B is characterized by a single nucleotide polymorphism (SNP) G560A (rs4986782...Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1. Drug Metab Rev 40, 169-184. Zheng, W., Deitz, A.C., Campbell

  13. The histone acetyltransferase MOF overexpression blunts cardiac hypertrophy by targeting ROS in mice.

    PubMed

    Qiao, Weiwei; Zhang, Weili; Gai, Yusheng; Zhao, Lan; Fan, Juexin

    2014-06-13

    Imbalance between histone acetylation/deacetylation critically participates in the expression of hypertrophic fetal genes and development of cardiac hypertrophy. While histone deacetylases play dual roles in hypertrophy, current evidence reveals that histone acetyltransferase such as p300 and PCAF act as pro-hypertrophic factors. However, it remains elusive whether some histone acetyltransferases can prevent the development of hypertrophy. Males absent on the first (MOF) is a histone acetyltransferase belonging to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. Here in this study, we reported that MOF expression was down-regulated in failing human hearts and hypertrophic murine hearts at protein and mRNA levels. To evaluate the roles of MOF in cardiac hypertrophy, we generated cardiac-specific MOF transgenic mice. MOF transgenic mice did not show any differences from their wide-type littermates at baseline. However, cardiac-specific MOF overexpression protected mice from transverse aortic constriction (TAC)-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observed lower expression of hypertrophic fetal genes in TAC-challenged MOF transgenic mice compared with that of wide-type mice. Mechanically, MOF overexpression increased the expression of Catalase and MnSOD, which blocked TAC-induced ROS and ROS downstream c-Raf-MEK-ERK pathway that promotes hypertrophy. Taken together, our findings identify a novel anti-hypertrophic role of MOF, and MOF is the first reported anti-hypertrophic histone acetyltransferase.

  14. Construction of a bioluminescent reporter strain to detect polychlorinated biphenyls

    SciTech Connect

    Layton, A.C.; Muccini, M.; Ghosh, M.M.; Sayler, G.S.

    1998-12-01

    A bioluminescent reporter strain, Ralstonia eutropha ENV307 (pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.

  15. Structure of the lac operon galactoside acetyltransferase.

    PubMed

    Wang, Xing-Guo; Olsen, Laurence R; Roderick, Steven L

    2002-04-01

    The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

  16. Evidence for arylamine N-acetyltransferase in Hymenolepis nana.

    PubMed

    Chung, J G; Kuo, H M; Wu, L T; Lai, J M; Lee, J H; Hung, C F

    1997-02-01

    N-acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Hymenolepis nana, a cestode found in the intestine of the Sprague-Dawley rats. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Hymenolepis nana whole tissue homogenizations were found to be 2.83 +/- 0.31 nmole/min/mg for 2-aminofluorene and 2.07 +/- 0.24 nmole/min/mg for p-aminobenzoic acid. The apparent Km and Vmax were 1.06 +/- 0.38 mM and 8.92 +/- 1.46 nmol/min/mg for 2-aminofluorene, and 2.16 +/- 0.19 mM and 12.68 +/- 2.26 nmol/min/mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide. At 0.25 mM iodacetamide the activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Fe2+, Ca2+ and Zn2+ were demonstrated to be the most potent inhibi-tors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetate, in contrast to other agents, markedly inhibited N-acetyltransferase activity. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in a cestode and extends the number of phyla in which this activity has been found.

  17. Construction and testing of orfA +/- FIV reporter viruses.

    PubMed

    Fadel, Hind J; Saenz, Dyana T; Poeschla, Eric M

    2012-01-01

    Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV), where the field has used genetically minimized transfer vectors with viral proteins supplied in trans. Here we report construction and use of a panel of single cycle FIV reporter viruses that express fluorescent protein markers. The viruses can be produced to high titer using human cell transfection and can transduce diverse target cells. To illustrate utility, we tested versions that are (+) and (-) for OrfA, an FIV accessory protein required for replication in primary lymphocytes and previously implicated in down-regulation of the primary FIV entry receptor CD134. We observed CD134 down-regulation after infection with or without OrfA, and equivalent virion production as well. These results suggest a role for FIV proteins besides Env or OrfA in CD134 down-regulation.

  18. Enhanced Geothermal Systems (EGS) well construction technology evaluation report.

    SciTech Connect

    Capuano, Louis, Jr.; Huh, Michael; Swanson, Robert; Raymond, David Wayne; Finger, John Travis; Mansure, Arthur James; Polsky, Yarom; Knudsen, Steven Dell

    2008-12-01

    Electricity production from geothermal resources is currently based on the exploitation of hydrothermal reservoirs. Hydrothermal reservoirs possess three ingredients critical to present day commercial extraction of subsurface heat: high temperature, in-situ fluid and high permeability. Relative to the total subsurface heat resource available, hydrothermal resources are geographically and quantitatively limited. A 2006 DOE sponsored study led by MIT entitled 'The Future of Geothermal Energy' estimates the thermal resource underlying the United States at depths between 3 km and 10 km to be on the order of 14 million EJ. For comparison purposes, total U.S. energy consumption in 2005 was 100 EJ. The overwhelming majority of this resource is present in geological formations which lack either in-situ fluid, permeability or both. Economical extraction of the heat in non-hydrothermal situations is termed Enhanced or Engineered Geothermal Systems (EGS). The technologies and processes required for EGS are currently in a developmental stage. Accessing the vast thermal resource between 3 km and 10 km in particular requires a significant extension of current hydrothermal practice, where wells rarely reach 3 km in depth. This report provides an assessment of well construction technology for EGS with two primary objectives: (1) Determining the ability of existing technologies to develop EGS wells. (2) Identifying critical well construction research lines and development technologies that are likely to enhance prospects for EGS viability and improve overall economics. Towards these ends, a methodology is followed in which a case study is developed to systematically and quantitatively evaluate EGS well construction technology needs. A baseline EGS well specification is first formulated. The steps, tasks and tools involved in the construction of this prospective baseline EGS well are then explicitly defined by a geothermal drilling contractor in terms of sequence, time and cost. A

  19. Final Report: Self Consolidating Concrete Construction for Modular Units

    SciTech Connect

    Gentry, Russell; Kahn, Lawrence; Kurtis, Kimberly; Petrovic, Bojan; Loreto, Giovanni; Van Wyk, Jurie; Canterero-Leal, Carlos

    2016-07-29

    This report outlines the development of a self-consolidating concrete (also termed “self-compacting concrete” or SCC) so that concrete placement can be made into steel plate composite (SC) modular structures without the need for continuous concrete placement. As part of the research, SCC mixtures were developed and validated to ensure sufficient shear capacity across cold-joints, while minimizing shrinkage and temperature increase during curing to enhance concrete bonding with the steel plate construction found in modular units. The self-roughening concrete produced as part of this research was assessed in SC structures at three scales: small-scale shear-friction specimens, mid-scale beams tested in in-plane and out-of-plane bending, and a full-scale validation test using an SC module produced by Westinghouse as part of the Plant Vogtle expansion. The experiments show that the self-roughening concrete can produce a cold-joint surface of 0.25 inches (6 mm) without external vibration during concrete placement. The experiments and subsequent analysis show that the shear friction provisions of ACI 318-14, Section 22.9 can be used to assess the shear capacity of the cold-joints in SC modular construction, and that friction coefficient of 1.35 is appropriate for use with these provisions.

  20. Arylamine N-Acetyltransferases in Mycobacteria

    PubMed Central

    Sim, Edith; Sandy, James; Evangelopoulos, Dimitrios; Fullam, Elizabeth; Bhakta, Sanjib; Westwood, Isaac; Krylova, Anna; Lack, Nathan; Noble, Martin

    2008-01-01

    Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure. PMID:18680471

  1. The Purification of Choline Acetyltransferase of Squid-Head Ganglia

    PubMed Central

    Husain, S. S.; Mautner, Henry G.

    1973-01-01

    Choline acetyltransferase (EC 2.3.1.6) isolated from the head ganglia of squid could be purified by use of mercurial-Sepharose columns as well as Sepharose columns to which the enzyme inhibitor p-(m-bromophenyl)vinyl pyridinium had been attached. These columns, in conjunction with 30-55% ammonium sulfate precipitation, 40-30% ammonium sulfate extraction, chromatography on sulfopropyl-Sephadex and on cellulose phosphate and hydroxylapatite columns, led to the isolation of three factions of choline acetyltransferase ranging in activity from 1000 to 4000 μmole/mg of protein/per hr. Polyacrylamide gel electrophoresis suggests that two of these fractions are homogeneous. The squid choline acetyltransferase is different from the mammalian-brain enzymes in having a larger molecular weight under the conditions used and in being relatively poorly inhibited by styryl pyridinium compounds. Images PMID:4521199

  2. Lift-Shape Construction, An EFL Project Report.

    ERIC Educational Resources Information Center

    Evans, Ben H.

    Research development of a construction system is detailed in terms of--(1) design and analysis, (2) construction methods, (3) testing, (4) cost analysis, and (5) architectural potentials. The system described permits construction of usual shapes without the use of conventional concrete formwork. The concrete involves development of a structural…

  3. Final Report: Self-Consolidating Concrete Construction for Modular Units

    SciTech Connect

    Gentry, Russell; Kahn, Lawrence; Kurtis, Kimberly; Petrovic, Bojan; Loreto, Giovanni; Van Wyk, Jurie; Canterero-Leal, Carlos

    2016-07-29

    This report focuses on work completed on DE-NE0000667, Self-Consolidating Concrete for Modular Units, in connection with the Department of Energy Nuclear Energy Enabling Technologies (DOE-NEET) program. This project was completed in the School of Civil and Environmental Engineering at the Georgia Institute of Technology, with Westinghouse Corporation as the industrial partner. The primary objective of this project was to develop self-consolidating concrete (also termed “self-compacting concrete” or SCC) mixtures so that concrete placement can be made into steel plate composite (SC) modular structures without the need for continuous concrete placement. As part of the research, SCC mixtures were developed and validated to ensure sufficient shear capacity across cold-joints, while minimizing shrinkage and temperature increase during curing to enhance concrete bonding with the steel plate construction found in modular units. The SCC mixtures developed were able to carry shearing forces across the cold-joint boundaries. This “self-roughening” was achieved by adding a tailored fraction of lightweight aggregate (LWA) to the concrete mix, some of which raised to the surface during curing, forming a rough surface on which subsequent concrete placements were made. The self-roughening behavior was validated through three sets of structural tests. Shear friction on small-scale specimens with cold joints was assessed using varying fractions of LWA and with varying amounts of external steel plate reinforcement. The results show that the shear friction coefficient, to be used with the provisions of ACI 318-14, Section 22.9, can be taken as 1.35. Mid-scale beam tests were completed to assess the cold-joint capacity in both in-plane and out-of-plane bending. The results showed that the self-roughened joints performed as well as monolithic joints. The final assessment was a full-scale test using a steel composite module supplied by Westinghouse and similar in construction to

  4. COMPONENTS FOR SCHOOL CONSTRUCTION IN THE MID-HUDSON REGION. FINAL REPORT 3.

    ERIC Educational Resources Information Center

    GREEN, ALAN D.; AND OTHERS

    THE FINAL REPORT OF A THREE PART FEASIBILITY STUDY OF THE COMPONENT CONSTRUCTION SYSTEMS SAMPLED APPROPRIATE ASPECTS OF THE SCHOOL CONSTRUCTION CLIMATE IN NEW YORK STATE. IT SOUGHT TO DETERMINE THE APPROPRIATENESS OF THE SCHOOL CONSTRUCTION SYSTEM DEVELOPEMENT PROCESS TO SCHOOL CONSTRUCTION IN THE MID-HUDSON VALLEY AND TO EXPLORE POSSIBILITIES FOR…

  5. Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferase encoded by the plasmid pSCS7 from Staphylococcus aureus.

    PubMed

    Schwarz, S; Cardoso, M

    1991-08-01

    The nucleotide sequence of the chloramphenicol acetyltransferase gene (cat) and its regulatory region, encoded by the plasmid pSCS7 from Staphylococcus aureus, was determined. The structural cat gene encoded a protein of 209 amino acids, which represented one monomer of the enzyme chloramphenicol acetyltransferase (CAT). Comparisons between the amino acid sequences of the pSCS7-encoded CAT from S. aureus and the previously sequenced CAT variants from S. aureus, Staphylococcus intermedius, Staphylococcus haemolyticus, Bacillus pumilis, Clostridium difficile, Clostridium perfringens, Escherichia coli, Shigella flexneri, and Proteus mirabilis were performed. An alignment of CAT amino acid sequences demonstrated the presence of 34 conserved amino acids among all CAT variants. These conserved residues were considered for their possible roles in the structure and function of CAT. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated relatively large evolutionary distances between the CAT variants of enteric bacteria, Clostridium, Bacillus, and Staphylococcus species.

  6. Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Franklin, K; Clarke, A J

    2001-08-01

    The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.

  7. [Construction and specificity of porcine bmp15 gene reporter vector].

    PubMed

    Qin, Mingming; Wei, Jianghua; Yu, Xiaoli; Zhang, Jinglong; Liu, Xiaopeng; Ma, Xiaoling; Wang, Huayan

    2014-02-01

    The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.

  8. USSR Report: Construction and Related Industries, No. 89.

    DTIC Science & Technology

    2007-11-02

    has arisen far from accidentally. Widespread use of high - strength concretes in construction is one of the main directions in technical policy for...cubic meters of high - strength reinforced concrete constructions. But today slightly more than 1 mil m3 are being pro- duced. In actuality, the...manufacture of high - strength constructions, as well as for low-grade concretes and mortars. In other words in either case the absence of a choice

  9. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  10. Early Returns: Tax Credit Bonds and School Construction? Policy Report.

    ERIC Educational Resources Information Center

    Mead, Sara

    A small federal program piloting tax credit bonds to support school construction, the Qualified Zone Academy Bond (QZAB), has existed since 1997--providing evidence of how tax credit bonds could work. This paper analyzes the results of QZABs to date in order to inform policymakers, advance the debate over federal school construction aid, and…

  11. Construction of relativistic quantum theory: a progress report

    SciTech Connect

    Noyes, H.P.

    1986-06-01

    We construct the particulate states of quantum physics using a recursive computer program that incorporates non-determinism by means of locally arbitrary choices. Quantum numbers and coupling constants arise from the construction via the unique 4-level combinatorial hierarchy. The construction defines indivisible quantum events with the requisite supraluminal correlations, yet does not allow supraluminal communication. Measurement criteria incorporate c, h-bar and m/sub p/ or (not ''and'') G, connected to laboratory events via finite particle number scattering theory and the counter paradigm. The resulting theory is discrete throughout, contains no infinities, and, as far as we have developed it, is in agreement with quantum mechanical and cosmological fact.

  12. COMPONENTS FOR SCHOOL CONSTRUCTION IN THE MID-HUDSON REGION. PROGRESS REPORT 2.

    ERIC Educational Resources Information Center

    HAVILAND, DAVID S.

    THIS STUDY IS PART 2 OF A PROGRESS REPORT ON A FEASIBILITY STUDY TO SAMPLE APPROPRIATE ASPECTS OF SCHOOL CONSTRUCTION FACTORS IN NEW YORK STATE. THE STUDY IS TO DETERMINE WHETHER THE SCHOOL CONSTRUCTION SYSTEMS DEVELOPMENT PROCESS IS APPLICABLE TO SCHOOL CONSTRUCTION IN THE MID-HUDSON VALLEY AND TO EXPLORE POSSIBLE MEANS OF ACHIEVING SUCH SCHOOL…

  13. COMPONENTS FOR SCHOOL CONSTRUCTION IN THE MID-HUDSON REGION. PROGRESS REPORT 1.

    ERIC Educational Resources Information Center

    GREEN, ALAN C.; AND OTHERS

    THIS REPORT IS PART 1 OF A FEASIBILITY STUDY TO SAMPLE APPROPRIATE ASPECTS OF THE SCHOOL CONSTRUCTION FACTORS IN NEW YORK STATE. THE STUDY IS TO DETERMINE WHETHER THE SCHOOL CONSTRUCTION SYSTEMS DEVELOPMENT PROCESS IS APPLICABLE TO SCHOOL CONSTRUCTION IN THE MID-HUDSON VALLEY. THE STUDY ALSO EXPLORES POSSIBLE MEANS BY WHICH SUCH SCHOOL BUILDINGS…

  14. 75 FR 75692 - Manufactured Home Construction and Safety Standards Act Reporting Requirements; Notice of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-06

    ... URBAN DEVELOPMENT Manufactured Home Construction and Safety Standards Act Reporting Requirements; Notice... Safety Standards Reporting Requirements. OMB Control Number, if applicable: 2502-0253. Description of the... Housing, HUD. ACTION: Notice. SUMMARY: The proposed information collection requirement described...

  15. High Life: 17th Annual Residence Hall Construction Report

    ERIC Educational Resources Information Center

    Agron, Joe

    2006-01-01

    Residence hall construction continues to be a priority for colleges and universities. With enrollments on the upswing, higher-education institutions are spending more and building larger facilities to entice students to live on campus. This article presents the findings of "American School & University's" 17th annual Residence Hall Construction…

  16. Artistic Judgment II: Construct Validation. Technical Report 1990-4.

    ERIC Educational Resources Information Center

    Bezruczko, Nikolaus; Schroeder, David H.

    The underlying constructs for an experimental battery (EB) consisting of artistic judgment tests--the Design Judgment Test (DJT), the Visual Designs Test (VDT), Proportion Appraisal (PA), and the Visual Aesthetic Sensitivity Test--were studied. Scores for 1,686 clients of the Johnson O'Connor Research Foundation's aptitude-testing service were…

  17. Report of CEC Study Committee on Construction Management.

    ERIC Educational Resources Information Center

    Consulting Engineers Council of the U.S., Washington, DC.

    Changing times place new demands on those involved in the implementation of construction projects. Within a relatively few years, the size and complexity of projects has grown substantially. Environmental and other public and social considerations are increasingly significant. With growing complexity, the requirements for effective project…

  18. Radioenzymatic assays for aminoglycosides with kanamycin 6'- acetyltransferase

    SciTech Connect

    Weber, A.; Smith, A.L.; Opheim, K.E.

    1985-03-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, the optimum conditions (pH, duration of incubation, and cofactor concentrations) were defined to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical standard curves at six different concentrations resulted in a correlation coefficient (r value) of greater than 0.99 for each aminoglycoside. The radioenzymatic assay correlates well with the bioassay (tobramycin and netilmicin) and radioimmunoassay (amikacin and kanamycin); the correlation coefficient is greater than 0.90 for all. The authors conclude that the radioenzymatic assay utilizing kanamycin 6'-acetyltransferase is feasible for all commercially available parenterally administered aminoglycosides.

  19. New perspectives for the regulation of acetyltransferase MOF.

    PubMed

    Li, Xiangzhi; Dou, Yali

    2010-04-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation.

  20. Special report on the audit of the management of Department of Energy construction projects

    SciTech Connect

    1996-11-21

    DOE`s FY 1996 budget of $18 billion included $1.1 billion for construction projects; ensuring that these projects meet bonafide existing or future DOE needs becomes increasingly important as DOE`s missions evolve and its organization changes. In 1994 and 1995, IG issued several reports expressing concerns about the construction planning process and questioned whether planned construction was necessary to meet mission needs. The reports also pointed out that DOE did not ensure that originally identified needs were still valid several years after a project`s conception. (The problems identified were at single locations.) While DOE management did not agree with all aspects of the audit reports, it canceled or downsized several projects and initiated a number of process improvements to enhance the construction planning process. Purpose of this report is to synthesize issues from these prior reports to assist management in focusing process improvement efforts to avoid construction of unneeded or oversized facilities.

  1. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  2. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  3. USSR Report, Construction and Related Industries, No. 99

    DTIC Science & Technology

    1983-11-10

    binding agent. We might add that slag may be used to make pumice, rubble, mineral wool — up to 15 types of materials and products for construct- ion...materials, large-size colored ceramic slabs is being manufactured. Technological lines have been created for the production of high-strength mineral ... wool slabs and asbestos slabs by the extrusion method, as well as plasterboard sheets of improved quality. However, as A. Yashin, minister of the

  4. Conceptual Design Report. Footprint Gallery Upgrade - Civil Construction, May 1988

    SciTech Connect

    1988-05-01

    The Footprint Gallery Complex will be enlarged and modified. The basic outline of the project will be to add 68,100 square feet of new construction, remodel 20,600 square feet of existing space, and retire by removal 17 ,500 square feet. The principal items to be addressed are: the creation of larger Main Control Rooms and Central Control Computer Rooms, the replacement of several temporary structures with permanent facilities, the provision for a growth in population of 132 people, and the creation of an intermediate sized meeting/lecture room facility. Disjointed second floor areas will be connected and made accessible to the handicapped, secure and informative viewing for visitors will be provided, and parking will be increased to match the expected growth. The new construction will provide for a more centralized concentration of systems and support personnel of the Fermilab Accelerator Division, reflecting the growth of these organizations during the last 15 years. Experiments, such as the D-Zero detector and antiproton deceleration (E760), have been assigned to the Accelerator Division for support. The associated physicists and experimenters make up the most significant component of the growth in population for which this construction will provide additional space.

  5. Construction

    DTIC Science & Technology

    2002-01-01

    Harbor Deepening Project, Jacksonville, FL Palm Valley Bridge Project, Jacksonville, FL Rotary Club of San Juan, San Juan, PR Tren Urbano Subway...David. What is nanotechnology? What are its implications for construction?, Foresight/CRISP Workshop on Nanotechnology, Royal Society of Arts

  6. Construction

    DTIC Science & Technology

    2002-01-01

    San Juan, PR Tren Urbano Subway Project, San Juan, PR U.S. Army South, San Juan, PR U.S. Coast Guard Housing Project, San Juan, PR U.S. Coast Guard...construction?, Foresight/CRISP Workshop on Nanotechnology, Royal Society of Arts . Cheltenham, England: 2001, p.5. 56 Concrete Proposals, Economist, July 24

  7. Construct Validity of Self-Reported Metacognitive Learning Strategies

    ERIC Educational Resources Information Center

    Berger, Jean-Louis; Karabenick, Stuart A.

    2016-01-01

    Despite their significant contributions to research on self-regulated learning, those favoring online and trace approaches have questioned the use of self-report to assess learners' use of learning strategies. An important rejoinder to such criticisms consists of examining the validity of self-report items. The present study was designed to assess…

  8. ALMA: status report on construction and early results from commissioning

    NASA Astrophysics Data System (ADS)

    Hills, Richard E.; Kurz, Richard J.; Peck, Alison B.

    2010-07-01

    The Atacama Large Millimeter/submillimeter Array (ALMA) is an international facility at an advanced stage of construction in the Atacama region of northern Chile. ALMA will consist of two arrays of high-precision antennas: one made up of twelve 7-meter diameter antennas operating in closely-packed configurations of about 50m in diameter, and the other of up to sixty-four 12-meter antennas arranged in configurations with diameters ranging from about 150 meters to 15 km. There will be four more 12-meter antennas to provide the "zero-spacing" information, which is critical for making accurate images of extended objects. The antennas will be equipped with sensitive millimeter-wave receivers covering most of the frequency range 84 to 950 GHz. State-of-the-art microwave, digital, photonic and software systems will capture the signals, transfer them to the central building and correlate them, while maintaining accurate synchronization. ALMA will provide images of a wide range of astronomical objects with great sensitivity and very high spectral resolution. The images will have much higher "fidelity" than those from existing mm/submm telescopes. This paper gives an update on the status of construction and on progress with the testing and scientific commissioning.

  9. Upper Sand Mountain Parish Solar Construction Workshops. Final performance report

    SciTech Connect

    Not Available

    1983-02-01

    The Upper Sand Mountain Parish continues to employ its initial strategy for involving high school vocational students with the pre cutting and instructional assembly aid to area families. The parish project works with high school vocational classes in pre fabbing solar devices into kit form. Then, students are employed to serve as instructors for Saturday construction workshops at the local electric cooperative. Trained teams of older and unemployed adults work with youth in building solar greenhouses for those able to pay labor. Over three years, the project has assisted and built 50 to 60 attached solar greenhouses with construction teams realizing in excess of $26,000 in labor for newly developed skills. The project continues to assist owners in monitoring and developing horticulturally as well as energy producing greenhouses. During the spring of 1982, the parish assisted greenhouse owners in marketing over 60,000 bedding plants worth over $3000. Monthly Greenhouse Owner Fellowship meetings have been a helpful setting for sharing of ideas and exchange of insights. A low interest solar loan fund, offering 5% loans for three years, has assisted over 30 families in going solar. The principle for this revolving fund has almost reached the $15,000 mark. The track record for loan repayments has been exceptional. Through workshops and tours we have aquainted hundreds of people across the southeast with low cost/low technology solar projects and a workable strategy for involving community groups and students in them. With church involvement, we have provided over $25,000 in grants to over 200 area families. Workshop information and plans are available to those interested for bread box solar water heaters, food dryers, window box collectors, insulation panels, and greenhouses.

  10. Fifth Annual Report: 2008 Pre-Construction Eelgrass Monitoring and Propagation for King County Outfall Mitigation

    SciTech Connect

    Woodruff, Dana L.; Judd, Chaeli; Thom, Ronald M.; Sather, Nichole K.; Kaufmann, Ronald M.

    2010-01-01

    This is the fifth and final report in a series documenting progress of the pre-construction eelgrass restoration and mitigation activities for the proposed King County Brightwater marine outfall, discharging to Puget Sound near Point Wells, Washington. King County began implementing a multiyear eelgrass monitoring and restoration program in 2004, with the primary goal of returning intertidal and shallow subtidal habitat and eelgrass to pre-construction conditions, after construction of the outfall. Major eelgrass mitigation program elements include: a) pre-construction monitoring, i.e., documenting initial eelgrass conditions and degree of fluctuation over a 5 year period prior to construction, b) eelgrass transplanting, including harvesting, offsite propagation and stockpiling of local plants for post-construction planting, and c) post-construction planting and subsequent monitoring, occurring in 2009 and beyond. The overall program is detailed in the Eelgrass Restoration and Biological Resources Implementation Workplan (King County 2008).

  11. An Examination of the Constructs Measured by Parent Behavioral Reports.

    ERIC Educational Resources Information Center

    Selden, J. J.; Pospisil, T. S.; Devaraju-Backhaus, S.; Bradley, J.; Michael, D. D.; Golden, C. J.

    Parents are often asked to complete behavioral checklists and personality inventories regarding their children when they bring them in for psychotherapy or neuropsychological testing. The Child Behavior Checklist (CBCL), the Conners' Parent Report Scale (CPRS), and the Personality Inventory for Children (PIC) are frequently used tests. Some…

  12. A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

    PubMed

    Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

    2015-04-01

    Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

  13. 35. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii. LOOKING BACK FROM STATION 335 AT RETURN CURVE. - Haleakala National Park Roads, Pukalani, Maui County, HI

  14. 36. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    36. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii. TYPICAL RUBBLE MASONRY HEADWALL AND BOX CULVERT. - Haleakala National Park Roads, Pukalani, Maui County, HI

  15. 38. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    38. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii. BRIDGE AT STATION 85+. - Haleakala National Park Roads, Pukalani, Maui County, HI

  16. Chemical biology of histone acetyltransferase natural compounds modulators.

    PubMed

    Piaz, Fabrizio Dal; Vassallo, Antonio; Rubio, Osmany Cuesta; Castellano, Sabrina; Sbardella, Gianluca; De Tommasi, Nunziatina

    2011-05-01

    Histone acetyltransferases (HATs) are a class of epigenetic enzymes crucial for chromatin restructuring and transcriptional regulation in eukaryotic cells, thus being a promising target for therapeutic development. Nonetheless, differently from histone deacetylases (HDACs) inhibitors, there is still paucity of small-molecule modulators of HAT activity. After a decline during past decade, natural products and their derivatives could be once again a valuable tool in the lead discovery process and meet such need of Novel Chemical Entities (NCEs). In this review, we will provide a comprehensive summary on the discovery of small-molecule HAT modulators from naturally occurring molecular scaffolds.

  17. Coherent constructive interference in Saturn's rings reported by Cassini VIMS

    NASA Astrophysics Data System (ADS)

    Nelson, R. M.; Hapke, B. W.; Brown, R. H.; Spilker, L. J.; Smythe, W. D.; Kamp, L.; Boryta, M.; Leader, F.; Matson, D. L.; Edgington, S.; Nicholson, P. D.; Filacchione, G.; Clark, R. N.; Bibring, J.-P.; Baines, K. H.; Buratti, B.; Bellucci, G.; Capaccioni, F.; Cerroni, P.; Combes, M.; Coradini, A.; Cruikshank, D. P.; Drossart, P.; Formisano, V.; Jaumann, R.; Langevin, Y.; McCord, T. B.; Mennella, V.; Sicardy, B.; Sotin, C.

    2007-08-01

    On several occasions during its orbital tour the Cassini spacecraft has flown between the sun and Saturn in such fashion that the zero phase point passed through the rings. The Visual and Infrared Mapping Spectrometer (VIMS) recorded spectral image cubes (0.4< ? <5.2μm) that showed the opposition effect (OE) at zero phase. The OE is a spike in the reflected light observed near 0o phase when it is displayed as a function of phase angle. This is the first time the OE has been resolved for small areas on the rings. Previous work has shown that the OE arises from two distinct processes, shadow hiding (SHOE) and coherent backscattering (CBOE). The SHOE process causes an OE by the elimination of shadows cast by regolith grains upon one another as phase angle decreases. The CBOE process causes an OE by constructive interference between photons traveling in opposite directions along the same path within the medium. SHOE is expected to dominate the contribution to the OE in absorbing media where multiple scattering of photons is not significant. CBOE is expected to dominate the contribution to the OE in highly reflective media with much multiple scattering. We have made spectral scans the VIMS images that traverse the zero phase point. We selected narrow spectral bands that reflected a variety of wavelengths and reflectance levels. In this way, phase curves of the ringlet were obtained for each band. We have compared these data to data we acquired in the laboratory using the JPL long arm goniometer where we measured the phase curve of particulate materials that simulate the surface of Saturn's ring particles. We argue here that the OE is due to coherent backscattering because: 1) The theoretical CBOE function fit to the data is excellent. 2) The OE width is extremely narrow 3) The angular width of the peak increases with wavelength. CBOE theory also predicts that the width depends on the transport mean free path (TMFP) in the medium. We find that the OE is caused by

  18. Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord.

    PubMed

    Soler-Botija, Carolina; Cuscó, Ivón; López, Eva; Clua, Agustín; Gich, Ignasi; Baiget, Montserrat; Ferrer, Isidre; Tizzano, Eduardo F

    2005-03-01

    We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.

  19. Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

    PubMed Central

    Lönnerberg, P; Lendahl, U; Funakoshi, H; Arhlund-Richter, L; Persson, H; Ibáñez, C F

    1995-01-01

    Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons. Images Fig. 1 Fig. 2 PMID:7732028

  20. The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

    PubMed Central

    Eberharter, Anton; Sterner, David E.; Schieltz, David; Hassan, Ahmed; Yates, John R.; Berger, Shelley L.; Workman, Jerry L.

    1999-01-01

    We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999–2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada− phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA. PMID:10490601

  1. Mixed Waste Management Facility (MWMF) closure, Savannah River Plant: Clay cap test section construction report

    SciTech Connect

    Not Available

    1988-02-26

    This report contains appendix 2 for the Clay Cap Test Section Construction Report for the Mixed Waste Management Facility (MWMF) closure at the Savannah River Plant. The Clay Cap Test Program was conducted to evaluate the source, Laboratory permeability, and compaction characteristics representative of Kaolin clays from the aiken, South Carolina vicinity. Included in this report are daily field reports Nos. 1 to 54. (KJD)

  2. Construction of Nationhood through Education in Malaya: Revisiting the Barnes and Fenn-Wu Reports

    ERIC Educational Resources Information Center

    Samuel, Moses; Khan, Mahmud Hasan

    2013-01-01

    This article provides an analysis of two colonial reports, the Barnes and the Fenn-Wu Reports on education in the British colony of Malaya. The popular stance on the Barnes and the Fenn-Wu Reports is that one is an effect or reply to the other. We argue on the contrary that the two reports construct a common argument on nation-building which…

  3. Executive summaries of reports leading to the construction of the Baca Geothermal Demonstration Project

    SciTech Connect

    Sherwood, P.B.; Newman, K.L.; Westermeier, J.F.; Giroux, H.D.; Lowe, G.D.; Nienberg, M.W.

    1980-05-01

    Executive summaries have been written for 61 reports and compilations of data which, in part, have led to the construction of the Baca 50 MW Geothermal Demonstration Project (GDP). The reports and data include environmental research, reservoir and feasibility studies, the project proposal to DOE and the Final Environmental Impact Statement. These executive summaries are intended to give the reader a general overview of each report prior to requesting the report from the GDP Data Manager.

  4. Executive summaries of reports leading to the construction of the Baca Geothermal Demonstration Project

    SciTech Connect

    Sherwood, P.B.; Newman, K.L.; Westermeier, J.F.; Giroux, H.D.; Lowe, G.D.; Nienberg, M.W.

    1980-05-01

    Executive summaries have been written for 61 reports and compilations of data which in part, have led to the construction of the Baca 50 MW Geothermal Demonstration Project (GDP). The reports and data include environmental research, reservoir and feasibility studies, the project proposal to DOE and the Final Environmental Impact Statement. These executive summaries are intended to give the reader a general overview of each report prior to requesting the report from the GDP Data Manager.

  5. Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

    PubMed

    Wapenaar, Hannah; van der Wouden, Petra E; Groves, Matthew R; Rotili, Dante; Mai, Antonello; Dekker, Frank J

    2015-11-13

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT.

  6. Enzyme kinetics and inhibition of histone acetyltransferase KAT8

    PubMed Central

    Wapenaar, Hannah; van der Wouden, Petra E.; Groves, Matthew R.; Rotili, Dante; Mai, Antonello; Dekker, Frank J.

    2016-01-01

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of AA was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  7. Structure and mechanism of non-histone protein acetyltransferase enzymes

    PubMed Central

    Friedmann, David R.

    2014-01-01

    Post translational modification (PTM) of proteins is ubiquitous and mediates many cellular processes including intracellular localization, protein-protein interactions, enzyme activity, transcriptional regulation and protein stability. While the role of phosphorylation as a key PTM has been well studied, the more evolutionarily conserved acetylation PTM has only recently attracted attention as a key regulator of cellular events. Protein acetylation has been largely studied in the context of its role in histone modification and gene regulation, where histones are modified by histone acetyltransferases (HATs) to promote transcription. However, more recent acetylomic and biochemical studies have revealed that acetylation is mediated by a broader family of protein acetyltransferases (PATs). The recent structure determination of several PATs has provided a wealth of molecular information regarding structural features of PATs, their enzymatic mechanisms, their mode of substrate-specific recognition and their regulatory elements. In this minireview, we will briefly describe what is known about non-histone protein substrates, but mainly focus on a few recent structures of PATs to compare and contrast them with HATs to better understand the molecular basis for protein recognition and modification by this burgeoning family of protein modification enzymes. PMID:23742047

  8. 47 CFR 17.57 - Report of radio transmitting antenna construction, alteration, and/or removal.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Report of radio transmitting antenna construction, alteration, and/or removal. 17.57 Section 17.57 Telecommunication FEDERAL COMMUNICATIONS... Marking and Lighting of Antenna Structures § 17.57 Report of radio transmitting antenna...

  9. 47 CFR 17.57 - Report of radio transmitting antenna construction, alteration, and/or removal.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 1 2013-10-01 2013-10-01 false Report of radio transmitting antenna construction, alteration, and/or removal. 17.57 Section 17.57 Telecommunication FEDERAL COMMUNICATIONS... Marking and Lighting of Antenna Structures § 17.57 Report of radio transmitting antenna...

  10. 47 CFR 17.57 - Report of radio transmitting antenna construction, alteration, and/or removal.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false Report of radio transmitting antenna construction, alteration, and/or removal. 17.57 Section 17.57 Telecommunication FEDERAL COMMUNICATIONS... Marking and Lighting of Antenna Structures § 17.57 Report of radio transmitting antenna...

  11. 47 CFR 17.57 - Report of radio transmitting antenna construction, alteration, and/or removal.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Report of radio transmitting antenna construction, alteration, and/or removal. 17.57 Section 17.57 Telecommunication FEDERAL COMMUNICATIONS... Marking and Lighting of Antenna Structures § 17.57 Report of radio transmitting antenna...

  12. 47 CFR 17.57 - Report of radio transmitting antenna construction, alteration, and/or removal.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false Report of radio transmitting antenna... COMMISSION GENERAL CONSTRUCTION, MARKING, AND LIGHTING OF ANTENNA STRUCTURES Specifications for Obstruction Marking and Lighting of Antenna Structures § 17.57 Report of radio transmitting antenna...

  13. Mixed Waste Management Facility (MWMF) closure, Savannah River Plant: Clay cap test section construction report

    SciTech Connect

    Not Available

    1988-02-26

    This report contains appendices 3 through 6 for the Clay Cap Test Section Construction Report for the Mixed Waste Management Facility (MWMF) closure at the Savannah River Plant. The Clay Cap Test Program was conducted to evaluate the source, lab. permeability, in-situ permeability, and compaction characteristics, representative of kaolin clays from the Aiken, South Carolina vicinity. (KJD)

  14. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  15. Structural and Functional Role of Acetyltransferase hMOF K274 Autoacetylation

    SciTech Connect

    McCullough, Cheryl E.; Song, Shufei; Shin, Michael H.; Johnson, F. Brad; Marmorstein, Ronen

    2016-07-05

    Many histone acetyltransferases undergo autoacetylation, either through chemical or enzymatic means, to potentiate enzymatic cognate substrate lysine acetylation, although the mode and molecular role of such autoacetylation is poorly understood. The MYST family of histone acetyltransferases is autoacetylated at an active site lysine residue to facilitate cognate substrate lysine binding and acetylation. Here, we report on a detailed molecular investigation of Lys-274 autoacetylation of the human MYST protein Males Absent on the First (hMOF). A mutational scan of hMOF Lys-274 reveals that all amino acid substitutions of this residue are able to bind cofactor but are significantly destabilized, both in vitro and in cells, and are catalytically inactive for cognate histone H4 peptide lysine acetylation. The x-ray crystal structure of a hMOF K274P mutant suggests that the reduced stability and catalytic activity stems from a disordering of the residue 274-harboring a α2-β7 loop. We also provide structural evidence that a C316S/E350Q mutant, which is defective for cognate substrate lysine acetylation; and biochemical evidence that a K268M mutant, which is defective for Lys-274 chemical acetylation in the context of a K274-peptide, can still undergo quantitative K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF Lys-274 autoacetylation in hMOF stability and cognate substrate acetylation and argues that binding of Ac-CoA to hMOF likely drives Lys-274 autoacetylation for subsequent cognate substrate acetylation.

  16. Site-specific methylated reporter constructs for functional analysis of DNA methylation.

    PubMed

    Han, Weiguo; Shi, Miao; Spivack, Simon D

    2013-11-01

    Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

  17. Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA

    PubMed Central

    Taschner, Michael; Vetter, Melanie; Lorentzen, Esben

    2012-01-01

    Acetylation of lysine residues is an important posttranslational modification found in all domains of life. α-tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on α-tubulin acetyltransferases. Here, we present the structure of the human α-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, α-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative α-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of α-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date. PMID:23071318

  18. A Plutonium Finishing Plant Model for the Cercla Removal Action and Decommissioning Construction Final Report

    SciTech Connect

    Hopkins, A.

    2008-07-01

    The joint policy between the U.S. Environmental Protection Agency (EPA) and the U.S. Department of Energy (DOE) for decommissioning buildings at DOE facilities documents an agreement between the agencies to perform decommissioning activities including demolition under the Comprehensive Environmental Response Compensation and Liability Act (CERCLA). The use of removal actions for decommissioning integrates EPA oversight authority, DOE lead agency responsibility, and state authority for decommissioning activities. Once removal actions have been performed under CERCLA, a construction completion report is required to document the completion of the required action. Additionally, a decommissioning report is required under DOE guidance. No direct guidance was found for documenting completion of decommissioning activities and preparing a final report that satisfies the CERCLA requirements and the DOE requirements for decommissioning. Additional guidance was needed for the documentation of construction completion under CERCLA for D and D projects undertaken under the joint policy that addresses the requirements of both agencies. A model for the construction completion report was developed to document construction completion for CERCLA D and D activities performed under the joint EPA/DOE policy at the Plutonium Finishing Plant (PFP). The model documentation report developed at PFP integrates the DOE requirements for establishing decommissioning end-points, documenting end-point completion and preparing a final decommissioning report with the CERCLA requirements to document completion of the action identified in the Action Memorandum (AM). The model includes the required information on health and safety, data management, cost and schedule and end-points completion. (authors)

  19. Homology modeling and identification of amino acids involved in the catalytic process of Mycobacterium tuberculosis serine acetyltransferase.

    PubMed

    Qiu, Juanjuan; Zang, Shizhu; Ma, Yufang; Owusu, Lawrence; Zhou, Lei; Jiang, Tao; Xin, Yi

    2017-03-01

    Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L‑cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti‑tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site‑directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti‑TB drugs.

  20. Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

    PubMed

    Gastel, J A; Roseboom, P H; Rinaldi, P A; Weller, J L; Klein, D C

    1998-02-27

    The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

  1. Assays for mechanistic investigations of protein/histone acetyltransferases.

    PubMed

    Berndsen, Christopher E; Denu, John M

    2005-08-01

    Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

  2. The MOZ histone acetyltransferase in epigenetic signaling and disease.

    PubMed

    Carlson, Samuel; Glass, Karen C

    2014-11-01

    The monocytic leukemic zinc finger (MOZ) histone acetyltransferase (HAT) plays a role in acute myeloid leukemia (AML). It functions as a quaternary complex with the bromodomain PHD finger protein 1 (BRPF1), the human Esa1-associated factor 6 homolog (hEAF6), and the inhibitor of growth 5 (ING5). Each of these subunits contain chromatin reader domains that recognize specific post-translational modifications (PTMs) on histone tails, and this recognition directs the MOZ HAT complex to specific chromatin substrates. The structure and function of these epigenetic reader modules has now been elucidated, and a model describing how the cooperative action of these domains regulates HAT activity in response to the epigenetic landscape is proposed. The emerging role of epigenetic reader domains in disease, and their therapeutic potential for many types of cancer is also highlighted.

  3. Generation of Driver and Reporter Constructs for the GAL4 Expression System in Drosophila.

    PubMed

    Southall, Tony D; Brand, Andrea H

    2008-07-01

    INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.

  4. Government Limitations on Training Innovations. A Construction Industry Cost Effectiveness Project Report. Report D-2.

    ERIC Educational Resources Information Center

    Business Roundtable, New York, NY.

    A study team researched impediments to the use of modern skill training methods in construction that have been caused by the U.S. Department of Labor. The problem that the study sought to define was whether the Labor Department impedes use of training innovations through the combined effect of the regulations promulgated by its Bureau of…

  5. Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

    SciTech Connect

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Martins, Marta; Ragunathan, Nilusha; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2007-10-01

    Bacillus anthracis arylamine N-acetyltransferase C (BanatC) is an enzyme that metabolizes the drug sulfamethoxazole. Crystals of the purified enzyme that diffract at 1.95 Å are reported. The arylamine N-acetyltransferase (NAT) enzymes are xenobiotic metabolizing enzymes that have been found in a large range of eukaryotes and prokaryotes. These enzymes catalyse the acetylation of arylamine drugs and/or pollutants. Recently, a Bacillus anthracis NAT isoform (BanatC) has been cloned and shown to acetylate the sulfonamide antimicrobial sulfamethoxazole (SMX). Subsequently, it was shown that BanatC contributes to the resistance of this bacterium to SMX. Here, the crystallization and the X-ray characterization of BanatC (Y38F mutant) are reported. The crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source.

  6. Mesoscopic tomography imaging of reporter genes in thick printed tissue constructs

    NASA Astrophysics Data System (ADS)

    Ozturk, Mehmet S.; Lee, Vivian K.; Zhao, Lingling; Dai, Guohoa; Intes, Xavier

    2013-06-01

    We report an application of Mesoscopic Fluorescence Molecular Tomography to 3D tissue engineering construct. Engineered thick tissue was hosting two 3D printed vasculatures. The channels were formed by live cells, expressing GFP and mCherry reporter genes, embedded in 3mm turbid media. Tissue and cells kept in a 3mm thick perfusion chamber during the entire imaging process which took less than 5 minutes.

  7. Non-Cognitive Constructs and Self-Reported Creativity by Domain

    ERIC Educational Resources Information Center

    Davis, Candice D.; Kaufman, James C.; McClure, Faith H.

    2011-01-01

    The purpose of this study was to determine the extent to which non-cognitive constructs (personality, thinking styles, motivation, and psychological well-being) would predict self-reported creativity across different domains among 266 college students. Consistent with hypotheses, openness, legislative thinking styles, and intrinsic motivation were…

  8. Mississippi's Annual Descriptive Report; Fiscal 1973, Under the Library Services and Construction Act.

    ERIC Educational Resources Information Center

    Mississippi Library Commission, Jackson.

    This report by the Mississippi Library Commission (MLC) describes its organization and activities and also library developments in Mississippi under the Library Services and Construction Act for the fiscal year 1973. The long range goals are listed as well as the resources development and services which the MLC has undertaken. The recent staff…

  9. Texas State Library: Library Services and Construction Act. Annual Report, FFY 1977.

    ERIC Educational Resources Information Center

    Texas State Library, Austin. Dept. of Library Development.

    Texas State Library activities for 1977 which were funded under the Library Services and Construction Act (LSCA) are reported, including administrative expenses for LSCA; administrative tape conversion--to duplicate books recorded on open reel tapes to cassettes for use by the blind; continuing education and consulting for librarians; special…

  10. A Report on the Design and Construction of the University of Massachusetts Computer Science Center.

    ERIC Educational Resources Information Center

    Massachusetts State Office of the Inspector General, Boston.

    This report describes a review conducted by the Massachusetts Office of the Inspector General on the construction of the Computer Science and Development Center at the University of Massachusetts, Amherst. The office initiated the review after hearing concerns about the management of the project, including its delayed completion and substantial…

  11. Identification, characterization and use of two tick promoters for construction of a dual luciferase reporter vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  12. 75 FR 62181 - Annual Materials Report on New Bridge Construction and Bridge Rehabilitation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-07

    ... Rehabilitation AGENCY: Federal Highway Administration (FHWA), DOT. ACTION: Notice. SUMMARY: Section 1114 of the... materials used in new Federal-aid bridge construction and bridge rehabilitation projects. As part of the... rehabilitation projects. Data on Federal-aid and non-Federal-aid highway bridges are included in the report...

  13. 40. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    40. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii, T.H. GENERAL VIEW OT THE PROJECT SHOWING CONSPICUOUS SCARS. THE BEFORE PHOTO OF A BEFORE AND AFTER SET. AFTER PHOTO IS HI-52-41. - Haleakala National Park Roads, Pukalani, Maui County, HI

  14. 39. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    39. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii, T.H., by Merel S. Sager, Resident Landscape Architect, April 16, 1935. COVERING CONSPICOUS ROCK FILLS WITH SOIL. - Haleakala National Park Roads, Pukalani, Maui County, HI

  15. 37. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    37. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii. HAND-LAID ROCK BERM ON RETURN CURVE TO PREVENT SCOUR AND SEEPAGE THROUGH FILLS. - Haleakala National Park Roads, Pukalani, Maui County, HI

  16. 34. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    34. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii, H.L. Handley, Assistant Highway Engineer, March 30, 1935. NOTE HOW THE LOCATION FITS THE CONTOUR OF THE HILL. LOOKING FROM STATION 382+00 ON HALEAKALA HIGHWAY. - Haleakala National Park Roads, Pukalani, Maui County, HI

  17. RADON REDUCTION AND RADON-RESISTANT CONSTRUCTION DEMONSTRATIONS IN NEW YORK - VOLUME 1: TECHNICAL REPORT

    EPA Science Inventory

    The report gives results of radon reduction and radon-resistant construction demonstrations in New York. The existing house evaluation demonstrated radon mitigation techniques where indoor radon concentrations exceeded 4 pCi/L. Results demonstrated that sealing all accessible fou...

  18. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences

    PubMed Central

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org. PMID:28004786

  19. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences.

    PubMed

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-12-22

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org.

  20. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization

    NASA Astrophysics Data System (ADS)

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-08-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer’s disease.

  1. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization

    PubMed Central

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer’s disease. PMID:27507101

  2. N-Acetyltransferase 1 Polymorphism and Breast Cancer Risk

    DTIC Science & Technology

    2009-10-01

    endonucleases, Apa I and Sph I (New England Biolabs, Ipswich, MA), followed by ligation with T4 Ligase (Invitrogen). Polyadenylation Site Removal NATa...ligated into pcDNA5/FRT using T4 ligase . In this report, these two constructs are referred to as NATa 1*4 and NATb 1*4. NATa and NATb NAT1*10, NAT1*11...NAT1*11 DNA was selected using restriction fragment length polymorphism (RFLP) and ligated into the vector using T4 ligase . NATa and NATb 1*14

  3. Canister storage building (CSB) safety analysis report phase 3: Safety analysis documentation supporting CSB construction

    SciTech Connect

    Garvin, L.J.

    1997-04-28

    The Canister Storage Building (CSB) will be constructed in the 200 East Area of the U.S. Department of Energy (DOE) Hanford Site. The CSB will be used to stage and store spent nuclear fuel (SNF) removed from the Hanford Site K Basins. The objective of this chapter is to describe the characteristics of the site on which the CSB will be located. This description will support the hazard analysis and accident analyses in Chapter 3.0. The purpose of this report is to provide an evaluation of the CSB design criteria, the design's compliance with the applicable criteria, and the basis for authorization to proceed with construction of the CSB.

  4. Generation IV Nuclear Energy Systems Construction Cost Reductions through the Use of Virtual Environments - Final Report

    SciTech Connect

    Timothy Shaw; Anthony Baratta; Vaughn Whisker

    2005-02-28

    Final report of 3 year DOE NERI-sponsored effort evaluating immersive virtual reality (CAVE) technology for design review, construction planning, and maintenance planning and training for next generation nuclear power plants. Program covers development of full-scale virtual mockups generated from 3D CAD data presented in a CAVE visualization facility. Mockups applied to design review of AP600/1000, Construction planning for AP 600, and AP 1000 maintenance evaluation. Proof of concept study also performed for GenIV PBMR models.

  5. Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator.

    PubMed

    Pegg, Anthony E

    2008-06-01

    Spermidine/spermine-N(1)-acetyltransferase (SSAT) regulates cellular polyamine content. Its acetylated products are either excreted from the cell or oxidized by acetylpolyamine oxidase. Since polyamines play critical roles in normal and neoplastic growth and in ion channel regulation, SSAT is a key enzyme in these processes. SSAT is very highly regulated. Its content is adjusted in response to alterations in polyamine content to maintain polyamine homeostasis. Certain polyamine analogs can mimic the induction of SSAT and cause a loss of normal polyamines. This may have utility in cancer chemotherapy. SSAT activity is also induced via a variety of other stimuli, including toxins, hormones, cytokines, nonsteroidal anti-inflammatory agents, natural products, and stress pathways, and by ischemia-reperfusion injury. These increases are initiated by alterations in Sat1 gene transcription reinforced by alterations at the other regulatory steps, including protein turnover, mRNA processing, and translation. Transgenic manipulation of SSAT activity has revealed that SSAT activity links polyamine metabolism to lipid and carbohydrate metabolism by means of alterations in the content of acetyl-CoA and ATP. A high level of SSAT stimulates flux through the polyamine biosynthetic pathway, since biosynthetic enzymes are induced in response to the fall in polyamines. This sets up a futile cycle in which ATP is used to generate S-adenosylmethionine for polyamine biosynthesis and acetyl-CoA is consumed in the acetylation reaction. A variety of other effects of increased SSAT activity include death of pancreatic cells, blockage of regenerative tissue growth, behavioral changes, keratosis follicularis spinulosa decalvans, and hair loss. These are very likely due to changes in polyamine and putrescine levels, although increased oxidative stress via the oxidation of acetylated polyamines may also contribute. Recently, it was found that the SSAT protein and/or a related protein, thialysine

  6. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    PubMed Central

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Cai-Hong

    2016-01-01

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity. PMID:27550639

  7. Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

    PubMed

    Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

    2017-02-10

    Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

  8. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    SciTech Connect

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Caihong

    2016-08-23

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.

  9. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    DOE PAGES

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; ...

    2016-08-23

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that maymore » contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.« less

  10. Biochemical characteristics of a novel vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa, Cymbopogon martinii var. Motia) leaf.

    PubMed

    Sharma, Pankaj K; Sangwan, Neelam S; Bose, Subir K; Sangwan, Rajender S

    2013-04-01

    Plants synthesize volatile alcohol esters on environmental insult or as metabolic induction during flower/fruit development. However, essential oil plants constitutively produce them as the oil constituents. Their synthesis is catalyzed by BAHD family enzymes called alcohol acyltransferases (AATs). However, no AAT has been characterized from plant foliage synthesizing acyclic monoterpenoids containing essential oils. Therefore, we have purified and biochemically characterized a geraniol: acetyl coenzyme A acetyltransferase (GAAT) from Palmarosa aroma grass (Cymbopogon martinii) leaf. MALDI-assisted proteomic study of the 43kDa monomeric enzyme revealed its sequence motif novelties e.g. relaxed conservation at Phe and Trp in DFGWG'. This suggests permissiveness of variations in the conserved motif without loss of catalytic ability. Also, some new conserved/semi-conserved motifs of AATs were recognized. The GAAT k(cat)/K(m) values (300-700M(-1)s(-1)) were low (a generic characteristic for secondary metabolism enzyme) but higher than those of some floral AATs. Wide substrate acceptability for catalyzing acetylation of diverse primary alcohols (chain of ≥C(6)) implied its catalytic description as a 'primary aliphatic alcohol acetyltransferase'. It signifies metabolic ability to deliver diverse aroma esters, should the acceptor alcohols be available in planta. To our knowledge, this is the first report of detailed kinetics of a vegetal monoterpenol acyltransferase.

  11. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    PubMed

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  12. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    PubMed Central

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  13. Innovation in Management of Primary School Construction: Multi-Purpose Primary School Buildings in Bangladesh. Educational Building Report 18.

    ERIC Educational Resources Information Center

    Constantinos, Fecadu

    This report deals with school building construction utilizing technology carried out by the Lutheran World Service/Rangpur Dinajpur Rehabilitation Service in Bangladesh. The purpose was to develop an alternative design for primary school constructions. The design, construction, and multipurpose use of the school buildings are described. Appended…

  14. Construction and testing of crumb rubber modified hot mix asphalt pavement. Interim report

    SciTech Connect

    Albritton, G.E.; Gatlin, G.R.

    1996-08-01

    This study was structured towards addressing that portion of ISTEA which directs the individual states to conduct studies on the recyclability of crumb rubber modified hot mix asphalt (CRMHMA), and the technical performance of CRMHMA pavement by monitoring the construction and evaluating the performance of highway test sections in which CRMHA is removed by cold milling and recycled into new HMA through a hot mix asphalt plant. This project is to be constructed in two phases, the CRMHMA will be built in the first phase and approximately one year later it will be recycled. This report deals with the first phase in which the objective was to further document the construction, engineering characteristics, and performace of CRMHMA.

  15. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  16. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii

    PubMed Central

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A.; Liu, Yunlong; Ferdig, Michael T.

    2016-01-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a 50% inhibitory concentration (IC50) similar to that for Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  17. In silico identification and characterization of N-Terminal acetyltransferase genes of poplar (Populus trichocarpa).

    PubMed

    Zhu, Hang-Yong; Li, Chun-Ming; Wang, Li-Feng; Bai, Hui; Li, Yan-Ping; Yu, Wen-Xi; Xia, De-An; Liu, Chang-Cai

    2014-01-27

    N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A-F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  18. Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.

    PubMed

    da Silva, Joas L; Piuri, Mariana; Broussard, Gregory; Marinelli, Laura J; Bastos, Gisele M; Hirata, Rosario D C; Hatfull, Graham F; Hirata, Mario H

    2013-07-01

    Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

  19. Obesity and lipid stress inhibit carnitine acetyltransferase activity[S

    PubMed Central

    Seiler, Sarah E.; Martin, Ola J.; Noland, Robert C.; Slentz, Dorothy H.; DeBalsi, Karen L.; Ilkayeva, Olga R.; An, Jie; Newgard, Christopher B.; Koves, Timothy R.; Muoio, Deborah M.

    2014-01-01

    Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. PMID:24395925

  20. Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

    PubMed

    Seiler, Sarah E; Koves, Timothy R; Gooding, Jessica R; Wong, Kari E; Stevens, Robert D; Ilkayeva, Olga R; Wittmann, April H; DeBalsi, Karen L; Davies, Michael N; Lindeboom, Lucas; Schrauwen, Patrick; Schrauwen-Hinderling, Vera B; Muoio, Deborah M

    2015-07-07

    Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

  1. Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

    PubMed Central

    Li, Yijia; Green, Keith D.; Johnson, Brooke R.

    2015-01-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn2+ metal ions displayed an inhibitory effect on the resistance enzyme AAC(6′)-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg2+, Cr3+, Cr6+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Au3+ with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2′)-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6′)-Ib′, AAC(6′)-Ie, AAC(6′)-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection. PMID:25941215

  2. Epigenetic Modulation using Small Molecules - Targeting Histone Acetyltransferases in Disease.

    PubMed

    Richters, André; Koehler, Angela N

    2017-02-23

    Histone acetyltransferases (HATs) are epigenetic drivers that catalyze the acetyl transfer from acetyl-CoA to lysines of both histone and non-histone substrates and thereby induce transcription either by chromatin remodeling or direct transcription factor activation. Histone deacetylases (HDACs) conduct the reverse reaction to counter HAT activity. Physiological processes such as cell cycle progression or apoptosis require a thoroughly balanced equilibrium of the interplay between acetylation and deacetylation processes to maintain or, if required, alter the global acetylome status. Aberrant HAT activity has recently been demonstrated to play a crucial role in the progression of various diseases such as prostate, lung, and colon cancers as well as glioblastomas and neurodegenerative diseases. Recent investigations have aimed for the identification of HAT modulators to further decipher the complexity of acetyl transferase related signaling cascades and discover potential leads for drug design approaches. HDACs have been extensively characterized and targeted by small molecules, including four FDA-approved HDAC inhibitors; in contrast, HATs have not been active targets for therapeutic development. This review will summarize the status of HAT associated diseases and the arsenal of currently known and available HAT inhibitors with respect to their discovery, further improvements, and current applications.

  3. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis.

    PubMed

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-14

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways.

  4. Autoacetylation of the MYST lysine acetyltransferase MOF protein.

    PubMed

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H; Neveu, John M; Zheng, Yujun George

    2012-10-12

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.

  5. Reconstruction of N-acetyltransferase 2 haplotypes using PHASE.

    PubMed

    Golka, Klaus; Blaszkewicz, Meinolf; Samimi, Mirabutaleb; Bolt, Hermann M; Selinski, Silvia

    2008-04-01

    The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2-4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).

  6. The histone acetyltransferase p300 promotes intrinsic axonal regeneration.

    PubMed

    Gaub, Perrine; Joshi, Yashashree; Wuttke, Anja; Naumann, Ulrike; Schnichels, Sven; Heiduschka, Peter; Di Giovanni, Simone

    2011-07-01

    Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.

  7. NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

    PubMed Central

    Berck, S.; Perret, X.; Quesada-Vincens, D.; Promé, J.-C.; Broughton, W. J.; Jabbouri, S.

    1999-01-01

    Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not. PMID:9922261

  8. Fourth Annual Report: 2007 Pre-Construction Eelgrass Monitoring and Propagation for King County Outfall Mitigation

    SciTech Connect

    Woodruff, Dana L.; Kohn, Nancy P.; Cullinan, Valerie I.; Southard, Susan S.; Vavrinec, John

    2007-10-04

    King County proposes to build a new sewer outfall discharging to Puget Sound near Point Wells, Washington. Construction is scheduled for 2008. The Point Wells site was selected to minimize effects on the nearshore marine environment, but unavoidable impacts to eelgrass (Zostera marina) beds are anticipated during construction. To mitigate these impacts and prepare for post-construction restoration, King County began implementing a multiyear eelgrass monitoring and restoration program in 2004, with the primary goal of returning intertidal and shallow subtidal habitat and eelgrass to pre-construction conditions. Major program elements related to eelgrass are (a) pre-construction monitoring, i.e., documenting initial eelgrass conditions and degree of fluctuation over 5 years prior to construction, (b) eelgrass transplanting, including harvesting, offsite propagating, and stockpiling of local plants for post-construction planting, and (c) post-construction planting and subsequent monitoring. The program is detailed in the Eelgrass Restoration and Biological Resources Implementation Workplan (King County 2006). This report describes calendar year 2007 pre-construction activities conducted by Pacific Northwest National Laboratory (PNNL) for King County. Activities included continued propagation of eelgrass shoots at the PNNL Marine Sciences Laboratory (MSL) in Sequim, Washington, and monitoring of the experimental harvest plots in the marine outfall corridor area to evaluate recovery rates relative to harvest rates. In addition, 490 eelgrass shoots were also harvested from the Marine Outfall Corridor in July 2007 to supplement the plants in the propagation tank at the MSL, bringing the total number of shoots to 1464. Eelgrass densities were monitored in four of five experimental harvest plots established in the Marine Outfall Corridor. Changes in eelgrass density were evaluated in year-to-year comparisons with initial harvest rates. A net increase in eelgrass density

  9. Final report: Constructing comprehensive models of grain boundaries using high-throughput experiments

    SciTech Connect

    Demkowicz, Michael; Schuh, Christopher; Marzouk, Youssef

    2016-08-29

    This is the final report on project DE-SC0008926. The goal of this project was to create capabilities for constructing, analyzing, and modeling experimental databases of the crystallographic characters and physical properties of thousands of individual grain boundaries (GBs) in polycrystalline metals. This project focused on gallium permeation through aluminum (Al) GBs and hydrogen uptake into nickel (Ni) GBs as model problems. This report summarizes the work done within the duration of this project (including the original three-year award and the subsequent one-year renewal), i.e. from August 1, 2012 until April 30, 2016.

  10. Third Annual Report: 2006 Pre-Construction Eelgrass Monitoring and Propagation for King County Outfall Mitigation

    SciTech Connect

    Woodruff, Dana L.; Southard, Susan S.; Cullinan, Valerie I.; Kohn, Nancy P.; Anderson, Michael G.; Vavrinec, John

    2007-02-01

    King County proposes to build a new sewer outfall discharging to Puget Sound near Point Wells, Washington. Construction is scheduled for 2008. The Point Wells site was selected to minimize effects on the nearshore marine environment, but unavoidable impacts to eelgrass (Zostera marina) beds are anticipated during construction. To mitigate for these impacts and prepare for post-construction restoration, King County began implementation of a multi-year eelgrass monitoring and restoration program in 2004, with the primary goal of returning intertidal and shallow subtidal habitat and eelgrass to pre-construction conditions. Major program elements are a) pre-construction monitoring, i.e., documenting initial eelgrass conditions and degree of fluctuation over 5 years prior to construction, b) eelgrass transplanting, including harvesting, offsite propagating and stockpiling of local plantstock, and post-construction planting, and c) post-construction monitoring. The program is detailed in the Eelgrass Restoration and Biological Resources Implementation Workplan (King County 2006). This report describes calendar year 2006 pre-construction activities conducted by Pacific Northwest National Laboratory (PNNL) in support of King County. Activities included continued propagation of eelgrass shoots and monitoring of the experimental harvest plots in the marine outfall corridor area to evaluate recovery rates relative to harvest rates. Approximately 1500 additional shoots were harvested from the marine outfall corridor in August 2006 to supplement the plants in the propagation tank at the PNNL Marine Sciences Laboratory in Sequim, Washington, bringing the total number of shoots to 4732. Eelgrass densities were monitored in the five experimental harvest plots established in the marine outfall corridor. Changes in eelgrass density were evaluated in year-to-year comparisons with initial harvest rates. Net eelgrass density decreased from 2004 post-harvest to 2006 in all plots

  11. Fungal Rtt109 Histone Acetyltransferase is an Unexpected Structural Homolog of Metazoan p300/CBP

    SciTech Connect

    Tang,Y.; Holbert, M.; Wurtele, H.; Meeth, K.; Rocha, W.; Gharib, M.; Jiang, E.; Thibault, P.; Verreault, A.; et al

    2008-01-01

    Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109-acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109-Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.

  12. Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

    PubMed

    Peach, C; Velten, J

    1992-02-01

    Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.

  13. Molecular Basis of Substrate Specific Acetylation by N-Terminal Acetyltransferase NatB.

    PubMed

    Hong, Haiyan; Cai, Yongfei; Zhang, Shijun; Ding, Hongyan; Wang, Haitao; Han, Aidong

    2017-04-04

    The NatB N-terminal acetyltransferase specifically acetylates the N-terminal group of substrate protein peptides starting with Met-Asp/Glu/Asn/Gln. How NatB recognizes and acetylates these substrates remains unknown. Here, we report crystal structures of a NatB holoenzyme from Candida albicans in the presence of its co-factor CoA and substrate peptides. The auxiliary subunit Naa25 of NatB forms a horseshoe-like deck to hold specifically its catalytic subunit Naa20. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. Moreover, a hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA. Together, these structures define a unique molecular mechanism of specific N-terminal acetylation acted by NatB.

  14. Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

    PubMed Central

    Perez-Leal, Oscar; Barrero, Carlos A.; Clarkson, Allen B.; Casero, Robert A.

    2012-01-01

    Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5′ stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity. PMID:22354986

  15. Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates

    SciTech Connect

    Hsiao,Y.; Jogl, G.; Tong, L.

    2006-01-01

    Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

  16. N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species.

    PubMed

    Du, Xiaoyi; Takagi, Hiroshi

    2007-07-01

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H(2)O(2), heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H(2)O(2) or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H(2)O(2). Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.

  17. Construct validity of a short, self report instrument assessing emotional dysregulation

    PubMed Central

    Powers, Abigail; Stevens, Jennifer; Fani, Negar; Bradley, Bekh

    2014-01-01

    There is a need for a brief measure of emotion dysregulation that can be used in large-scale studies. This study evaluated the construct validity of a short, self-report instrument of emotion dysregulation. Subjects (N=2197) were recruited from primary care clinics of an urban public hospital as part of a study of trauma-related risk and resilience. Emotion dysregulation was measured using the Emotion Dysregulation Scale, short version (EDS-short), a12-item self-report measure assessing emotional experiencing, cognition, and behavior. EDS-short was first compared with the Difficulties in Emotion Regulation Scale (DERS). Then, the construct validity of the EDS-short in predicting depression, posttraumatic stress, substance abuse, borderline pathology, suicide attempts, psychiatric hospitalizations, positive affect, and resiliency was assessed. We found a significant positive correlation between EDS-short and DERS. The EDS-short was significantly predictive of higher reported depressive, posttraumatic stress, substance abuse, and borderline symptoms, and lower reported positive affect and resiliency, over and above demographic characteristics and negative affect. Our results demonstrate that the EDS-short is a useful instrument for measuring emotion dysregulation in traumatized populations. A brief measure of emotion dysregulation is critical as the field moves forward in studying the wide ranging negative effects of emotion dysregulation across psychiatric disorders and outcomes. PMID:25468625

  18. Environmental assessment report: Nuclear Test Technology Complex. [Construction and operation of proposed facility

    SciTech Connect

    Tonnessen, K.; Tewes, H.A.

    1982-08-01

    The US Department of Energy (USDOE) is planning to construct and operate a structure, designated the Nuclear Test Technology Complex (NTTC), on a site located west of and adjacent to the Lawrence Livermore National Laboratory. The NTTC is designed to house 350 nuclear test program personnel, and will accommodate the needs of the entire staff of the continuing Nuclear Test Program (NTP). The project has three phases: land acquisition, facility construction and facility operation. The purpose of this environmental assessment report is to describe the activities associated with the three phases of the NTTC project and to evaluate potential environmental disruptions. The project site is located in a rural area of southeastern Alameda County, California, where the primary land use is agriculture; however, the County has zoned the area for industrial development. The environmental impacts of the project include surface disturbance, high noise levels, possible increases in site erosion, and decreased air quality. These impacts will occur primarily during the construction phase of the NTTC project and can be mitigated in part by measures proposed in this report.

  19. Histone acetyltransferases: challenges in targeting bi-substrate enzymes.

    PubMed

    Wapenaar, Hannah; Dekker, Frank J

    2016-01-01

    Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to neurological disorders, both through acetylations of histone proteins and non-histone proteins. Several HAT inhibitors, like bi-substrate inhibitors, natural product derivatives, small molecules, and protein-protein interaction inhibitors, have been developed. Despite their potential, a large gap remains between the biological activity of inhibitors in in vitro studies and their potential use as therapeutic agents. To bridge this gap, new potent HAT inhibitors with improved properties need to be developed. However, several challenges have been encountered in the investigation of HATs and HAT inhibitors that hinder the development of new HAT inhibitors. HATs have been shown to function in complexes consisting of many proteins. These complexes play a role in the activity and target specificity of HATs, which limits the translation of in vitro to in vivo experiments. The current HAT inhibitors suffer from undesired properties like anti-oxidant activity, reactivity, instability, low potency, or lack of selectivity between HAT subtypes and other enzymes. A characteristic feature of HATs is that they are bi-substrate enzymes that catalyze reactions between two substrates: the cofactor acetyl coenzyme A (Ac-CoA) and a lysine-containing substrate. This has important-but frequently overlooked-consequences for the determination of the inhibitory potency of small molecule HAT inhibitors and the reproducibility of enzyme inhibition experiments. We envision that a careful characterization of molecular aspects of HATs and HAT inhibitors, such as the HAT catalytic mechanism and the enzyme kinetics of small molecule HAT inhibitors, will greatly improve the development of potent and

  20. Mixed Waste Management Facility (MWMF) closure, Savannah River Plant: Clay cap test section construction report

    SciTech Connect

    Not Available

    1988-02-26

    This report summarizes the information gathered in constructing the clay cap test section. The purpose of the test section was to determine compaction characteristics of four representative kaolin clays and demonstrate in-situ permeability for these clays of 1 {times} 10 {sup {minus}7} cm/sec or less. The final technical specifications with regard to maximum clod size, acceptable ranges of placement water content, lift thickness, and degree of compaction will be based on experience gained from the test section. The data derived from this study will also be used in the development of Quality Assurance (QA) and Quality Control (QC) methods to be used during actual cap construction of the Mixed Waste Management Facility (MWMF) Closure project. 7 tabs.

  1. H-coal pilot plant. Phase II. Construction. Phase III. Operation. Annual report No. 3

    SciTech Connect

    Not Available

    1981-02-04

    At the request of DOE Oak Ridge, ASFI agreed to assume responsibility for completion of Plant construction in December, 1979, at which time Badger Plants' on-site work was ended. This construction effort consisted of electric heat tracing and insulation of piping and instrumentation. At the close of the reporting period the work was completed, or was projected to be completed, within the ASFI budgeted amounts and by dates that will not impact Plant operations. Engineering design solutions were completed for problems encountered with such equipment as the High Pressure Letdown Valves; Slurry Block Valves; Slurry Pumps; the Bowl Mill System; the Dowtherm System; and the Ebullating Pump. A Corrosion Monitoring Program was established. With the exception of Area 500, the Antisolvent Deashing Unit, all operating units were commissioned and operated during the reporting period. Coal was first introduced into the Plant on May 29, 1980, with coal operations continuing periodically through September 30, 1980. The longest continuous coal run was 119 hours. A total of 677 tons of Kentucky No. 11 Coal were processed during the reporting period. The problems encountered were mechanical, not process, in nature. Various Environmental and Health programs were implemented to assure worker safety and protection and to obtain data from Plant operations for scientific analysis. These comprehensive programs will contribute greatly in determining the acceptability of long term H-Coal Plant operations.

  2. Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

    PubMed Central

    Liu, Boyin; Bossing, Torsten

    2016-01-01

    We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently. PMID:27725692

  3. Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells.

    PubMed Central

    Giantini, M; Shatkin, A J

    1989-01-01

    The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3. Images PMID:2724407

  4. 41. From Final Construction Report on the Haleakala Road ProjectNR7, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    41. From Final Construction Report on the Haleakala Road Project--NR-7, Hawaii National Park, Island of Maui, Territory of Hawaii, T.H. VIEW FROM APPROXIMATELY THE SAME SPOT SHOWING HOW COVERING THE ROCK FILLS WITH SOIL HAS ALMOST OBLITERATED THESE SCARS. TO IDENTIFY A POINT FOR COMPARISON NOTICE THE BRIDE GULCH JUST TO THE LEFT OF THE CENTER IN THE UPPER PICTURE AND COMPARE WITH THE SAME GULCH IN THE LOWER PICTURE. THE AFTER PHOTO OF A BEFORE AND AFTER SET. BEFORE PHOTO IS HI-52-40. - Haleakala National Park Roads, Pukalani, Maui County, HI

  5. Evaluating the Construct-Coverage of the e-rater[R] Scoring Engine. Research Report. ETS RR-09-01

    ERIC Educational Resources Information Center

    Quinlan, Thomas; Higgins, Derrick; Wolff, Susanne

    2009-01-01

    This report evaluates the construct coverage of the e-rater[R[ scoring engine. The matter of construct coverage depends on whether one defines writing skill, in terms of process or product. Originally, the e-rater engine consisted of a large set of components with a proven ability to predict human holistic scores. By organizing these capabilities…

  6. 33 CFR 148.415 - When conducting site evaluation and pre-construction testing, what must be reported?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) Within 120 days after the site evaluation or pre-construction testing, a final written report must be... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false When conducting site evaluation... Site Evaluation and Pre-Construction Testing § 148.415 When conducting site evaluation and...

  7. Final audit report of remedial action construction at the UMTRA Project Falls City, Texas, site

    SciTech Connect

    1995-05-01

    This final audit report for the Falls City, Texas, Uranium Mill Tailings Remedial Action Project site summarizes the radiological audits and the quality assurance (QA) in-process surveillances, audits, and final close-out inspection performed by the U.S. Department of Energy (DOE) and Technical Assistance Contractor (TAC). It also summarizes U.S. Nuclear Regulatory Commission (NRC) surveillances. One radiological audit and three radiological surveillances were performed at the Falls City site. These surveillances and audit, which resulted in 31 observations, focused primarily on processing site activities and were performed on the following dates: 3-6 August 1992, 29-30 October 1992, 22-26 March 1993, and 1-3 November 1993. All outstanding radiological issues were closed out at the completion of the construction activities. Six QA in-process surveillances, which resulted in 71 observations, were performed at the Falls City site on the following dates: 22-24 July 1992, 23-25 November 1992, 17-19 May 1993, 16-18 August 1993, 13-15 October 1993, and 2-4 February 1994. All outstanding issues were closed out with the February surveillance on 3 March 1994. The DOE/TAC remedial action close-out inspections of the Falls City site, which resulted in 56 observations, were conducted 9-10 June 1994 and 26 July 1994. The inspections were closed out on 26 January 1995. The NRC performed three on-site construction reviews (OSCR), resulting in seven observations of remedial action construction activities that occurred during site visits. The OSCRs were performed 9 December 1992, 12 May 1993, and 25 October 1993. Since all audit and surveillance observations and recommendations have been closed out, this final audit report segment of the site certification process is complete.

  8. Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls.

    PubMed

    Bailey, Jason A; Lahiri, Debomoy K

    2012-08-01

    Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that Ch

  9. A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

    PubMed Central

    Ohba, Reiko; Steger, David J.; Brownell, James E.; Mizzen, Craig A.; Cook, Richard G.; Côté, Jacques; Workman, Jerry L.; Allis, C. David

    1999-01-01

    Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. PMID:10022893

  10. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  11. Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.

    PubMed

    Shimizu, Takeshi; Ohta, Yuko; Tsutsuki, Hiroyasu; Noda, Masatoshi

    2011-06-01

    A novel chromosome-plasmid hybrid bioluminescent reporter system (C-P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia coli (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C-P reporter system is useful for determining the gene expression profile of bacteria.

  12. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  13. Conditions for the self-catalysed inactivation of carnitine acetyltransferase. A novel form of enzyme inhibition

    PubMed Central

    Chase, J. F. A.; Tubbs, P. K.

    1969-01-01

    1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(−)-carnitine plus CoA or of bromoacetyl-CoA plus (−)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromoacetyl substrate analogue; in each case about 1mole of inhibitor is required to inactivate completely 1mole of enzyme of molecular weight 58000±3000. 3. Inhibition by bromoacetyl-CoA plus (−)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (−)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (−)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4°. 5. Incubation with S-carboxymethyl-CoA (−)-carnitine ester causes a slow inhibition of carnitine acetyltransferase. 6. The formation of bound S-carboxymethyl-CoA (−)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme–substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase. PMID:5763788

  14. Genetic Variation at the N-acetyltransferase (NAT) Genes in Global Populations

    EPA Science Inventory

    Functional variability at the N-acetyltransferase (NAT) genes is associated with adverse drug reactions and cancer susceptibility in humans. Previous studies of small sets of ethnic groups have indicated that the NAT genes have high levels of amino acid variation that differ in f...

  15. Phylogenetic and biological investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and eukaryotic organisms. The role of NATs in fungal biology has only recently been investigated. The NAT1 (FDB2) gene of Fusarium verticillioides was the first NAT cloned and character...

  16. AAC(3)-XI, a New Aminoglycoside 3-N-Acetyltransferase from Corynebacterium striatum

    PubMed Central

    Galimand, Marc; Fishovitz, Jennifer; Lambert, Thierry; Barbe, Valérie; Zajicek, Jaroslav

    2015-01-01

    Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI. PMID:26149994

  17. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    PubMed

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

  18. Comparative investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated. The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fun...

  19. The facC Gene of Aspergillus nidulans Encodes an Acetate-Inducible Carnitine Acetyltransferase

    PubMed Central

    Stemple, Christopher J.; Davis, Meryl A.; Hynes, Michael J.

    1998-01-01

    Mutations in the facC gene of Aspergillus nidulans result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the facC gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in facC mutants, while fatty acid-inducible activity was absent in an acuJ mutant. Acetate induction of facC expression was dependent on the facB regulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′ facC sequences. Carbon catabolite repression of facC expression was affected by mutations in the creA gene and a CreA fusion protein bound to 5′ facC sequences. Mutations in the acuJ gene led to increased acetate induction of facC expression and also of an amdS-lacZ reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of facB. A model is presented in which facC encodes a cytosolic CAT enzyme, while a different CAT enzyme, which is acuJ dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments. PMID:9829933

  20. Design report small-scale fuel alcohol plant. Volume II. Detailed construction information

    SciTech Connect

    Not Available

    1980-12-01

    The objectives of the report are to (a) provide potential alcohol producers with a reference design and (b) provide a complete, demonstrated design of a small-scale fuel alcohol plant. This report describes a small-scale fuel alcohol plant designed and constructed for the DOE by EG and G Idaho, Inc., an operating contractor at the Idaho National Engineering Laboratory. The plant is reasonably complete, having the capability for feedstock preparation, cooking, saccharification, fermentation, distillation, by-product dewatering, and process steam generation. An interesting feature is an instrumentation and control system designed to allow the plant to run 24 hours per day with only four hours of operator attention. Where possible, this document follows the design requirements established in the DOE publication Fuel From Farms, which was published in February 1980. For instance, critical requirements such as using corn as the primary feedstock, production of 25 gallons of 190 proof ethanol per hour, and using batch fermentation were taken from Fuel From Farms. One significant deviation is alcohol dehydration. Fuel From Farms recommends the use of a molecular sieve for dehydration, but a preliminary design raised significant questions about the cost effectiveness of this approach. A cost trade-off study is currently under way to establish the best alcohol dehydration method and will be the subject of a later report. Volume two includes equipment and instrumentation data sheets, instrument loop wiring diagrams, and vendor lists.

  1. Unc-5 homolog B (UNC5B) is one of the key downstream targets of N-α-Acetyltransferase 10 (Naa10)

    PubMed Central

    Xu, Huiyu; Han, Yong; Liu, Bing; Li, Rong

    2016-01-01

    N-α-acetyltransferase 10 (Naa10) displays alpha (N-terminal) acetyltransferase activity. It functions as a major modulator of cell growth and differentiation. Until now, a few downstream targets were found, but no studies have concerned about which gene is the early event of Naa10 downstream target. As we know, the earlier events may play more significant role in Naa10 pathway. Through construction of Naa10 stably knocked down H1299 cell line, we discovered cell morphological changes induced by Naa10. Moreover, potential function of Naa10 in cell morphogenesis was also indicated using cDNA microarray analysis of the Naa10 stably knock-down cell line. We further discovered that netrin-1 (NTN1) and its receptor UNC-5 Homology B (UNC5B) were the early event among the genes involved in Naa10 stably knocked down induced genes expression changes in cell morphogenesis. This was further validated in caudal half region of E10 mouse embryos. Negative regulation of Naa10 towards NTN1 and its receptor UNC5B were also detected upon treatment of all-trans retinoid acid, which was often used to induce morphological differentiation. PMID:27910960

  2. Design and construction of the NMSU Geothermally Heated Greenhouse Research Facility: Final technical report

    SciTech Connect

    Schoenmackers, R.

    1988-11-01

    This report describes the design, construction, and performance of the New Mexico State University (NMSU) Geothermal Greenhouse Research Facility. Two 6000-square-foot greenhouses were built on the NMSU campus and supplied with geothermal energy for heating. The geothermal water is pumped from one of three wells producing water at temperatures from 141/degree/F to 148/degree/F. Heat is delivered to the greenhouse space by means of overhead fan-coil unit heaters. The two greenhouses are double-glazed on roof and wall surfaces employing a total of four different film materials: Tedlar/Reg Sign/, Melinex/Reg Sign/, Softglass/Reg Sign/, and Agrifilm/Reg Sign/. One greenhouse is cooled using a traditional fan and pad cooling system. The second greenhouse is cooled with a high-pressure fog system and natural ventilation through roof and side vents. A 2400-square-foot metal building next to the greenhouses provides office, work, and storage space for the facility. The greenhouse facility was leased to two commerical tenants who produced a variety of crops. The performance of the greenhouses was monitored and reported both qualitatively and quantitatively. Results from the tenant's pilot-scale studies in the NMSU greenhouse facility were transferred and applied to two commercial greenhouse ranges that were built in southern New Mexico during 1986/87. 9 figs., 5 tabs.

  3. Interim radon-resistant construction guidelines for use in Florida-1989. Final report, August 1987-June 1988

    SciTech Connect

    Pugh, T.D.

    1990-08-01

    The report gives results of a project to investigate, analyze, and develop radon-resistant construction guidelines that are consistent with other building codes and that could be applied to Florida. A literature search resulted in information on radon remediation techniques, new construction methods, and existing radon-resistant building codes such as those in Sweden and Canada. The identified techniques were amended, modified, or supplemented for incorporation into draft model guidelines for new construction in format consistent with the Southern Building Code Congress International, Inc.'s Standard Building Code. A technical review advisory committee formed during development of the guidelines, provided input and recommended changes to the draft guidelines. Although the project has resulted in guidelines for recommended construction practices, they should be coupled with a carefully planned and implemented program of experimentation. Eventually, this approach will lead to building code provisions that are scientifically defensible, cost effective, reliable, and easily incorporated into standard construction practice.

  4. Space station system analysis study. Part 3: Documentation. Volume 2: Technical report. [structural design and construction

    NASA Technical Reports Server (NTRS)

    1977-01-01

    An analysis of construction operation is presented as well as power system sizing requirements. Mission hardware requirements are reviewed in detail. Space construction base and design configurations are also examined.

  5. 42 CFR 137.353 - What is contained in a construction project financial report?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... HUMAN SERVICES INDIAN HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES TRIBAL SELF-GOVERNANCE Construction Roles of Self-Governance Tribe in Establishing and Implementing Construction Project Agreements... 42 Public Health 1 2010-10-01 2010-10-01 false What is contained in a construction...

  6. 42 CFR 137.352 - What is contained in a construction project progress report?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... HUMAN SERVICES INDIAN HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES TRIBAL SELF-GOVERNANCE Construction Roles of Self-Governance Tribe in Establishing and Implementing Construction Project Agreements... 42 Public Health 1 2010-10-01 2010-10-01 false What is contained in a construction...

  7. Closure Report for Corrective Action Unit 392: Spill Sites and Construction Materials, Nevada Test Site, Nevada

    SciTech Connect

    R. B. Jackson

    2002-02-01

    This Closure Report documents the closure activities that were conducted to close Corrective Action Unit (CAU) 392--Spill Sites and Construction Materials located on the Nevada Test Site (NTS). CAU 392 is listed on in Appendix III of the Federal Facility Agreement and Consent Order (FFACO) (FFACO, 1996) and consists of the following six Corrective Action Sites (CASs) located in Areas 5 and 6 of the NTS: CAS 05-17-02 Construction Materials/Lead Bricks; CAS 06-17-03 Cement Mud Pit; CAS 06-1 9-01 Cable Pile; Powder Piles (3); CAS 06-44-02 Paint Spill; CAS 06-44-03 Plaster Spill; CAS 06-44-04 Cutting Fluid Discharge Ditch. Closure activities were performed in two phases. Phase 1 activities consisted of collecting waste characterization samples of soil and material present on-site, and where appropriate, performing radiological screening of debris at the six CASs. Results were used to determine how waste generated during closure activities would be handled and disposed of, i.e., as nonhazardous sanitary or hazardous waste, etc. Phase 2 activities consisted of closing each CAS by removing debris and/or soil, disposing of the generated waste, and verifying that each CAS was clean closed by visual inspection and/or by the collecting soil verification samples for laboratory analysis. Copies of the analytical results for the site verification samples are included in Appendix A. Copies of the Sectored Housekeeping Site Closure Verification Form for each of the six CASs are included in Appendix 8. Appendix C contains a copy of the Bechtel Nevada (BN) On-site Waste Transport Manifest for the hazardous waste generated during closure of CAS 06-44-02.

  8. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

    PubMed Central

    2010-01-01

    Background In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal

  9. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  10. Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

    SciTech Connect

    Lau, E Y; Felton, J S; Lightstone, F C

    2006-06-06

    A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.

  11. Evaluation Criteria for Musculoskeletal and Craniofacial Tissue Engineering Constructs: A Conference Report

    PubMed Central

    2008-01-01

    Over the past 20 years, tissue engineering (TE) has evolved into a thriving research and commercial development field. However, applying TE strategies to musculoskeletal (MSK) and craniofacial tissues has been particularly challenging since these tissues must also transmit loads during activities of daily living. To address this need, organizers invited a small group of bioengineers, surgeons, biologists, and material scientists from academia, industry, and government to participate in a 2½-day conference to develop general and tissue-specific criteria for evaluating new concepts and tissue-engineered constructs, including threshold values of success. Participants were assigned to four breakout groups representing commonly injured tissues, including tendon and ligament, articular cartilage, meniscus and temporomandibular joint, and bone and intervertebral disc. Working in multidisciplinary teams, participants first carefully defined one or two important unmet clinical needs for each tissue type, including current standards of care and the potential impact of TE solutions. The groups then sought to identify important parameters for evaluating repair outcomes in preclinical studies and to specify minimally acceptable values for these parameters. The importance of in vitro TE studies was then discussed in the context of these preclinical studies. Where data were not currently available from clinical, preclinical, or culture studies, the groups sought to identify important areas of preclinical research needed to speed the development process. This report summarizes the findings of the conference. PMID:19093294

  12. Conceptual Design Report: Fermilab Upgrade: Main Injector - Technical Components and Civil Construction, January 1990 (Rev. 2)

    SciTech Connect

    none,

    1990-01-10

    This report contains a description of the design and cost estimate of a new 150 GeV accelerator, designated the Main Injector, which will be required to support the upgrade of the Fermilab Accelerator Complex. The construction of this accelerator will simultaneously result in significant enhancements to both the Fermilab collider and fixed target programs. The Main Injector (MI) is to be located south of the Antiproton Source and tangent to the Tevatron ring at the FO straight section as shown in Figure 1-1. The MI will perform all duties currently required of the existing Main Ring. Thus, operation of the Main Ring will cease following commissioning of the MI, with a concurrent reduction in background rates as seen in the colliding beam detectors. The performance of the MI, as measured in terms of protons per second delivered to the antiproton production target or total protons delivered to the Tevatron, is expected to exceed that of the Main Ring by a factor of two to three. In addition the MI will provide high duty factor 120 GeV beam to the experimental areas during collider operation, a capability which does not presently exist in the Main Ring.

  13. The chromosomal 2'-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation.

    PubMed

    Macinga, D R; Rather, P N

    1999-02-01

    Intrinsic chromosomal acetyltransferases involved in aminoglycoside resistance have been identified in a number of bacteria. In Providencia stuartii, a chromosomal acetyltransferase (AAC(2')-Ia) has been characterized in detail. In addition to the ability to acetylate aminoglycosides, the AAC(2')-Ia enzyme has at least one physiological function, which is the acetylation of peptidoglycan. This modification is likely to influence the autolytic system in P. stuartii. The regulation of aac(2')-Ia expression is extremely complex involving at least seven regulatory genes acting in at least two pathways. This complexity in regulation indicates that aac(2')-Ia expression must be tightly controlled in response to different environmental conditions. This presumably reflects the importance of maintaining correct levels of peptidoglycan acetylation. In this review, a summary of data will be presented involving both the physiological and genetic aspects of aac(2')-Ia in P. stuartii.

  14. Resistance to apramycin in two enterobacterial clinical isolates: detection of a 3-N-acetyltransferase IV.

    PubMed

    Gómez-Lus, R; Rivera, M J; Gómez-Lus, M L; Gil, J; Gómez-Lus, S; Castillo, J; Goñi, P; Madero, P; Rubio, M C

    1990-08-01

    Considering the possible role of farm animals in the contamination of human consumers by plasmid-mediated apramycin-resistant enterobacteria strains, this type of resistance should be tested more systematically in human isolates. Very recently we isolated in Zaragoza one apramycin-resistant Escheria coli strain obtained from the blood of a hospitalized patient; this clinical isolate produced a plasmid-mediated 3-N-aminoglycoside acetyltransferase IV. We describe also the isolation in Madrid of one multiresistant Klebsiella pneumoniae clinical strain. This isolate harbored a single plasmid and carried determinants for apramycin, gentamicin, tobramycin, hygromycin B, streptomycin, and ampicillin, which could be transferred en bloc to E. coli K-12 J62. Extracts from donor and transconjugant strains carrying pUZ6776 plasmid produce acetyltransferase activity AAC(3)-IV and double phosphotransferase activity (HPH and APH(3'')).

  15. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

    PubMed Central

    2010-01-01

    Background Histone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene. PMID:20096118

  16. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    SciTech Connect

    Maes, Dominique Crabeel, Marjolaine; Van de Weerdt, Cécile; Martial, Joseph; Peeters, Eveline; Charlier, Daniël; Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid

    2006-12-01

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.

  17. Strength and consolidation characteristics of coal refuse for design and construction of disposal facilities: Applications of research findings. Final report

    SciTech Connect

    Huang, Y.H.

    1987-08-01

    The use of coal refuse properties for the short-term stability analysis of disposal facilities is illustrated in the report. Three types of disposal facilities are considered: waste embankments constructed of combined or fine refuse, refuse dams constructed by upstream method, and sandwich construction by placing coarse and fine refuse in alternate layers. Detailed procedures are presented to determine the factor of safety at the end of construction using the REAME computer program developed at the University of Kentucky. The results of analysis show that limiting the maximum moisture content of combined or fine refuse in waste embankments and keeping the construction of refuse dams at a slower pace are very important for short-term stability. Due to the granular nature of coarse and fine refuse, the use of sandwich construction appears to be an efficient and economical disposal method. To compare the properties of coal refuse from the Western Coal Field with those from the Eastern Coal Field reported previously, test results on combined and fine refuse taken from two sites in Colorado are presented.

  18. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    SciTech Connect

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.

  19. Radon reduction and radon-resistant construction demonstrations in New York. Final report, September 1986-June 1988

    SciTech Connect

    Nitschke, I.

    1989-01-01

    The report covers three tasks related to indoor radon: (1) the demonstration of radon reduction techniques in 8 houses in each of two uniquely different radon prone areas of the State of New York; (2) the evaluation and repair of 14 radon mitigation systems in houses mitigated 4 years earlier; and (3) the development and application of radon resistant new construction designs in 15 different houses. In applying radon reduction techniques in existing houses, techniques which were applicable in extremely porous soil were not as easily applied to houses built on granite ledge; combinations of techniques may be required in many difficult houses before an acceptable radon level can be achieved. During the study, basement pressurization was applied as a radon reduction technique for the first time. The radon resistant new construction designs should demonstrate effective methods of sealing out radon during construction; however, quality control problems prevalent in the construction industry may require additional laboratory tests for verification.

  20. Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

    PubMed Central

    Hintermann, E; Grieder, N C; Amherd, R; Brodbeck, D; Meyer, U A

    1996-01-01

    In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8901578

  1. Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad*

    PubMed Central

    Kubiak, Xavier; Li de la Sierra-Gallay, Inès; Chaffotte, Alain F.; Pluvinage, Benjamin; Weber, Patrick; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2013-01-01

    Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms. PMID:23770703

  2. Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana.

    PubMed

    Boycheva, Irina; Vassileva, Valya; Revalska, Miglena; Zehirov, Grigor; Iantcheva, Anelia

    2017-03-01

    In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacterium-mediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle.

  3. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus: differences from other mycobacterial isoforms and implications for selective inhibition.

    PubMed

    Cocaign, Angélique; Kubiak, Xavier; Xu, Ximing; Garnier, Guillaume; Li de la Sierra-Gallay, Inès; Chi-Bui, Linh; Dairou, Julien; Busi, Florent; Abuhammad, Areej; Haouz, Ahmed; Dupret, Jean Marie; Herrmann, Jean Louis; Rodrigues-Lima, Fernando

    2014-11-01

    Mycobacterium abscessus is the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies of M. abscessus proteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamine N-acetyltransferase (NAT) from M. abscessus [(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significant N-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid and p-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smooth M. abscessus morphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related to Nocardia farcinica NAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance of M. abscessus.

  4. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  5. MOZ and MORF acetyltransferases: Molecular interaction, animal development and human disease.

    PubMed

    Yang, Xiang-Jiao

    2015-08-01

    Lysine residues are subject to many forms of covalent modification and one such modification is acetylation of the ε-amino group. Initially identified on histone proteins in the 1960s, lysine acetylation is now considered as an important form of post-translational modification that rivals phosphorylation. However, only about a dozen of human lysine acetyltransferases have been identified. Among them are MOZ (monocytic leukemia zinc finger protein; a.k.a. MYST3 and KAT6A) and its paralog MORF (a.k.a. MYST4 and KAT6B). Although there is a distantly related protein in Drosophila and sea urchin, these two enzymes are vertebrate-specific. They form tetrameric complexes with BRPF1 (bromodomain- and PHD finger-containing protein 1) and two small non-catalytic subunits. These two acetyltransferases and BRPF1 play key roles in various developmental processes; for example, they are important for development of hematopoietic and neural stem cells. The human KAT6A and KAT6B genes are recurrently mutated in leukemia, non-hematologic malignancies, and multiple developmental disorders displaying intellectual disability and various other abnormalities. In addition, the BRPF1 gene is mutated in childhood leukemia and adult medulloblastoma. Therefore, these two acetyltransferases and their partner BRPF1 are important in animal development and human disease.

  6. Histone H3 specific acetyltransferases are essential for cell cycle progression

    PubMed Central

    Howe, LeAnn; Auston, Darryl; Grant, Patrick; John, Sam; Cook, Richard G.; Workman, Jerry L.; Pillus, Lorraine

    2001-01-01

    Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G2/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities. PMID:11731478

  7. 77 FR 53251 - Annual Materials Report on New Bridge Construction and Bridge Rehabilitation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-31

    ... Rehabilitation AGENCY: Federal Highway Administration (FHWA), DOT. ACTION: Notice. SUMMARY: Section 1114 of the... used in new Federal-aid bridge construction and bridge rehabilitation projects. As part of the SAFETEA... bridge construction and bridge rehabilitation projects. Data on Federal-aid and non-Federal-aid...

  8. Innovation in Construction of Small Secondary Schools in Thailand. Educational Building Report 19.

    ERIC Educational Resources Information Center

    Ministry of Education, Bangkok (Thailand).

    In 1987, a project was initiated in Thailand to develop a prototype to be used as a standard design for the construction of rural secondary schools in all areas of the country. The project had three objectives: (1) to derive architectural designs for classroom and auxiliary buildings that could be constructed within available budgets and would be…

  9. Self-Constructs and Anxiety Across Cultures. Research Report. ETS RR-09-12

    ERIC Educational Resources Information Center

    Lee, Jihyun

    2009-01-01

    This study examined the factorial structure of three related constructs, math self-concept, math self-efficacy, and math anxiety, across 41 countries. One factorial structure was achieved at both between- and within-country levels. Within-country variations of the self-constructs were also noted in relation to math performance: Self-concept showed…

  10. Field performance of maintenance treatments constructed with reclaimed asphalt pavement (RAP). Final research report, September 1992-August 1994

    SciTech Connect

    Estakhri, C.K.

    1994-11-01

    In the study, RAP was blended with recycling emulsions and conventional maintenance mixtures in attempts to improve its field performance as a maintenance mixture. RAP was also mixed with stabilizers and used as a base material in maintenance projects. Several field experiments were constructed throughout the state, and the report documents their performance.

  11. Construct Validation of Analytic Rating Scales in a Speaking Assessment: Reporting a Score Profile and a Composite

    ERIC Educational Resources Information Center

    Sawaki, Yasuyo

    2007-01-01

    This is a construct validation study of a second language speaking assessment that reported a language profile based on analytic rating scales and a composite score. The study addressed three key issues: score dependability, convergent/discriminant validity of analytic rating scales and the weighting of analytic ratings in the composite score.…

  12. Brief Report: Preliminary Reliability, Construct Validity and Standardization of the Auditory Behavior Questionnaire (ABQ) for Children with Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    Egelhoff, Kelsey; Lane, Alison E.

    2013-01-01

    The Auditory Behavior Questionnaire (ABQ) evaluates abnormal behavioral responses to auditory stimulation in children with Autism Spectrum Disorder (ASD). This study reports preliminary reliability, construct validity and standardization of the ABQ. Parents of children with ASD aged 7-21 years (n = 165) completed the ABQ on-line. Cronbach's alpha…

  13. The Jackson State University Honors Dormitory: An Evaluation of Design, Construction, and Maintenance. Report to the Mississippi Legislature.

    ERIC Educational Resources Information Center

    2000

    This report assesses: (1) whether the Jackson State University (JSU) Honors Dormitory required nearly $1 million in repairs two years after it was built because of inappropriate design, construction, and maintenance; and (2) if the state or university has legal recourse to recover damages resulting from inferior design and/or construction…

  14. 10 CFR 51.50 - Environmental report-construction permit, early site permit, or combined license stage.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... costs of the proposed action or an evaluation of alternative energy sources. (3) For other than light... 10 Energy 2 2014-01-01 2014-01-01 false Environmental report-construction permit, early site permit, or combined license stage. 51.50 Section 51.50 Energy NUCLEAR REGULATORY COMMISSION...

  15. 10 CFR 51.50 - Environmental report-construction permit, early site permit, or combined license stage.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... costs of the proposed action or an evaluation of alternative energy sources. (3) For other than light... 10 Energy 2 2011-01-01 2011-01-01 false Environmental report-construction permit, early site permit, or combined license stage. 51.50 Section 51.50 Energy NUCLEAR REGULATORY COMMISSION...

  16. 10 CFR 51.50 - Environmental report-construction permit, early site permit, or combined license stage.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... costs of the proposed action or an evaluation of alternative energy sources. (3) For other than light... 10 Energy 2 2012-01-01 2012-01-01 false Environmental report-construction permit, early site permit, or combined license stage. 51.50 Section 51.50 Energy NUCLEAR REGULATORY COMMISSION...

  17. 10 CFR 51.50 - Environmental report-construction permit, early site permit, or combined license stage.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... costs of the proposed action or an evaluation of alternative energy sources. (3) For other than light... 10 Energy 2 2010-01-01 2010-01-01 false Environmental report-construction permit, early site permit, or combined license stage. 51.50 Section 51.50 Energy NUCLEAR REGULATORY COMMISSION...

  18. 10 CFR 51.50 - Environmental report-construction permit, early site permit, or combined license stage.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... costs of the proposed action or an evaluation of alternative energy sources. (3) For other than light... 10 Energy 2 2013-01-01 2013-01-01 false Environmental report-construction permit, early site permit, or combined license stage. 51.50 Section 51.50 Energy NUCLEAR REGULATORY COMMISSION...

  19. Indonesia--Innovation in the Management of Primary School Construction: A Case Study. Education Building Report 8.

    ERIC Educational Resources Information Center

    Hussin

    This UNESCO report describes the progress of primary school building development under the second Five Year Plan of the Government of Indonesia. The main objective of the construction program was to increase the enrollement of children of primary school age to 85 per cent of all eligible children. Chapter I provides an historical perspective on…

  20. Conceptual Design Report: Fermilab Main Injector - Technical Components and Civil Construction, April 1992 (Rev. 3.1)

    SciTech Connect

    None, None

    1992-04-01

    This report contains a description of the design and cost estimation of a new 150 GeV accelerator, designated the Fermilab Main Injector (FMI). The construction of this accelerator will simulataneously result in significant enhancements to both the Fermilab collider and fixed target programs.

  1. Measuring Teacher Self-Report on Classroom Practices: Construct Validity and Reliability of the Classroom Strategies Scale-Teacher Form

    ERIC Educational Resources Information Center

    Reddy, Linda A.; Dudek, Christopher M.; Fabiano, Gregory A.; Peters, Stephanie

    2015-01-01

    This article presents information about the construct validity and reliability of a new teacher self-report measure of classroom instructional and behavioral practices (the Classroom Strategies Scales-Teacher Form; CSS-T). The theoretical underpinnings and empirical basis for the instructional and behavioral management scales are presented.…

  2. The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition

    PubMed Central

    Huang, Fu; Saraf, Anita; Florens, Laurence; Kusch, Thomas; Swanson, Selene K.; Szerszen, Leanne T.; Li, Ge; Dutta, Arnob; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2016-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. Here, we report the identification of subunits of the Drosophila Enok complex and demonstrate that all subunits are important for its HAT activity. We further report a novel interaction between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, our study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin. PMID:27198229

  3. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.

    PubMed

    Lilly, M; Lambrechts, M G; Pretorius, I S

    2000-02-01

    The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid

  4. Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli

    PubMed Central

    Dusthackeer, Azger; Kumar, Vanaja; Subbian, Selvakumar; Sivaramakrishnan, Gomathi; Zhu, Guofang; Subramanyam, Balaji; Hassan, Sameer; Nagamaiah, Selvakumar; Chan, John; Rama, Narayanan Paranji

    2008-01-01

    The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha-crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern. PMID:18272245

  5. Writers and Their Maps: The Construction of a GAO Report on Sexual Harassment.

    ERIC Educational Resources Information Center

    Cook, Kelli Cargile

    2000-01-01

    Examines a 1994 General Accounting Office (GAO) report on sexual harassment at U.S. service academies to determine how power structures affected the report writers' rhetorical choices. Identifies what is valued and devalued in the report's contents. Describes Congress's reaction to the report and speculates on the report's impact on public…

  6. Detailed project plan: Design, construction and operation of pilot scale Charfuel{reg_sign} process. Topical report, Task 2

    SciTech Connect

    Not Available

    1993-09-01

    In this project, a pilot-scale facility for the flash hydropyrolysis of coal will be designed, built and operated to demonstrate the integrated operation of critical components of the CHARFUEL process and to obtain scale-up data for subsequent demonstration facility for the production of a clean coal slurry fuel. This report presents project plans which includes detailed construction plan; procurement of materials and equipment; construction, test and start-up; potential problems and solutions during operations; data collection and analysis; and feasibility analysis.

  7. Distal tibial physeal bridge: a complication from a tension band plate and screw construct. Report of a case.

    PubMed

    Oda, Jon E; Thacker, Mihir M

    2013-05-01

    We report on a case of a tension band plate and screw construct (Eight Plate) used over the anterior distal tibia in an 9-year-old girl in an attempt to induce recurvatum of the ankle joint to correct a recalcitrant equinus deformity. With growth of the distal tibial physis, the epiphyseal screw was drawn through the physis into the distal tibial metaphysis, resulting in the creation of a transphyseal bony bar. Caution should be exercised when attempting temporary hemiepiphyseodesis using a plate and screw construct in small epiphyses or in an osteopenic bone.

  8. Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation1,2,3

    PubMed Central

    Wee, Caroline L.; Teo, Shaun; Oey, Nicodemus E.; Wright, Graham D.; VanDongen, Hendrika M.A.

    2014-01-01

    Abstract Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the β-spectrin isoform βSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and βSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation. PMID:26464963

  9. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase

    SciTech Connect

    Leblanc, G.C.; Trimmer, B.A.; Landis, S.C.

    1987-05-01

    Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. The authors report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat measured by radioimmunoassay. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what had been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase. These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues.

  10. New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

    SciTech Connect

    Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K.

    2015-10-15

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

  11. APIC state-of-the-Art report: the role of infection control during construction in health care facilities.

    PubMed

    Bartley, J M

    2000-04-01

    The Association for Professionals in Infection Control and Epidemiology, Inc (APIC), is a multidisciplinary organization of more than 12,000 health care professionals who practice infection control and epidemiology within a variety of health care settings. This report reviews issues the infection control professional should consider related to construction and renovation projects in health care facilities. Preventing transmission of infectious agents to vulnerable patient populations, health care workers, and visitors remains an important component of infection control programs. Environmental dispersal of microorganisms during construction, resulting in nosocomial infections, has been described previously, and select examples are provided in Table I as a reminder that there is a solid, scientific basis for these concerns. Environmental airborne contaminants and infectious agents are closely related to water and moisture-related conditions and figure prominently in construction activity. Weems et al have established construction activity as an independent variable for infectious risks in such circumstances. Construction-related outbreak literature will not be revisited in detail; however, pertinent citations will identify resources as appropriate.

  12. Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania.

    PubMed

    Folgueira, Cristina; Requena, Jose M

    2007-10-01

    Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.

  13. TRW/ORE-IDA potato-processing project: construction phase. Final report

    SciTech Connect

    Cherne, J; Logan, J

    1981-07-23

    A solar process heat system has been installed at an existing potato processing plant in Oregon. After a brief description of the location, commercial hardware, predicted performance and contracting procedures, the system is described subsystem-by-subsystem, including the parabolic trough collector field, steam generator, freeze prevention, computerized control system, data acquisition system, and various ancillary equipment. The operating modes are discussed, including normal operation, freeze prevention, control, and data acquisition operation. The construction process and problems encountered during construction and start-up are discussed. A paper on the control scheme and the data acquisition system functional specification are appended. A set of 23 record drawings illustrates the system. (LEW)

  14. 30 MJ superconducting magnetic energy storage stabilizing coil. Final report for construction

    SciTech Connect

    1983-03-01

    This report covers Phase II, Fabrication and Delivery of the 30 MJ Superconducting Magnetic Energy Storage Stabilizing Coil. A history of the manufacturing and assembly phase of the magnet is presented. Major problems and solutions are summarized, and illustrations of the major operations are provided. The Quality Assurance program is described with a listing of all nonconformance reports. Design documentation is provided, including a Design Document Index, monthly progress reports, and a list of papers given on the project. Appendices to the report contain copies of released and revised design calculations, test reports, assembly procedure, and nonconformance reports and engineering dispositions.

  15. Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

    PubMed Central

    Ikeda, Keiko; Steger, David J.; Eberharter, Anton; Workman, Jerry L.

    1999-01-01

    Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions. PMID:9858608

  16. Urban School Construction: A Case Study of Alternative Financing Methods for St. Louis, Missouri. A Report.

    ERIC Educational Resources Information Center

    Fitzgibbon, James; And Others

    The authors, after discussing the St. Louis school system and its financial history, survey both traditional and innovative construction finance alternatives that have been used across the country. These alternatives, which fall into two categories, include: (1) conventional financing through tax incomes including State and Federal aid, and (2)…

  17. The Laborers-AGC Construction Skills Training Program. Final Performance Report.

    ERIC Educational Resources Information Center

    Tippie, John L.; Rice, Eric

    Patterned after a previously successful Laborers-Associated General Contractors model named the Construction Skills Training Program, a demonstration project was implemented at five regional training centers. At least eight courses were created, combined, or revised. Four full-length audiovisual support pieces were completed. Three courses were…

  18. Human factors in the management of Becon Construction Company's Heavy Oil Test Station Project. Final report

    SciTech Connect

    Maurer, D.

    1986-11-01

    In January 1986, the Becon Construction Company - an open-shop contractor based in Houston, Texas - mobilized a work force to a construction project location at the Belridge Oil Field, near Bakersfield, California. The construction craftsmen and helpers in Becon's work force were expected to improve their level of productivity as the work proceeded because of the knowledge and skills that they would acquire as a result of the repetitive nature of the work. As it was originally organized, the Becon HOTS project represented almost a pure laboratory environment in which to collect data concerning the learning effect on worker productivity as well as to statistically isolate the impact on productivity of such external factors as weather, absenteeism, turnover, and especially work methods improvement techniques and pay incentives. From the outset of the HOTS project, the project manager had established an accurate system to quantitatively measure and compare the total manhours that each crew - civil, mechanical, electrical, and prefabrication -- performed at individual HOTS construction locations. Additionally, it was initially assumed that the project manager would be relatively free to implement changes to the job site conditions involving incentive pay and work methods improvement. The subsequent effect of these changes on the productivity of separate work crews could then be evaluated in terms of measured manhours per crew per HOTS.

  19. Cyclone-Resistant Rural Primary School Construction - A Design Guide. Educational Building Report 7.

    ERIC Educational Resources Information Center

    Sinnamon, Ian T.; Loo, G. A. van't

    One of a series of documents on design of disaster-resistant buildings, this publication treats construction of rural primary schools to resist destruction by wind and water from cyclones. Also appropriate for other buildings, material is aimed at rural primary schools because they are less likely to be professionally designed or supervised; the…

  20. A Formal Construction of Term Classes. Technical Report No. TR73-18.

    ERIC Educational Resources Information Center

    Yu, Clement T.

    The computational complexity of a formal process for the construction of term classes for information retrieval is examined. While the process is proven to be difficult computationally, heuristic methods are applied. Experimental results are obtained to illustrate the maximum possible improvement in system performance of retrieval using the formal…

  1. Teacher-Constructed Frames for Instruction with Content Area Text. Technical Report No. 537.

    ERIC Educational Resources Information Center

    Armstrong, James O.; And Others

    An exploratory study developed ways to describe systematically a particular graphic organizer, the frame. For the study, 27 middle-grade teachers each constructed 2 frames on sections of fourth-grade science and social studies texts. Teachers also worked collaboratively in pairs and larger groups to produce frames. Collaborative sessions were…

  2. Innovation in Primary School Construction: Maldives Community Schools. Educational Building Report 12.

    ERIC Educational Resources Information Center

    Luthfi, Mohamed; Zubair, Habeeba

    The Maldives experience of constructing community schools is examined to show how other small countries may overcome the special problems faced in designing and building schools. External assistance for design, and training of national personnel qualified neither as architects or engineers, was used to make up for the lack of a permanent unit for…

  3. Higher Order Characterization of Heuristics for Compass and Straight Edge Constructions in Geometry. Report No. 70.

    ERIC Educational Resources Information Center

    Scandura, Joseph M.; And Others

    A quasi-systematic strategy of devising rule sets for problem solving is applied to ruler and compass geometrical constructions. "Lower order" rules consisting of basic skills and "higher order" rules which govern the selection and combination of lower order rules are identified by an analysis of problem types; three types of…

  4. Item Pool Construction Using Mixed Integer Quadratic Programming (MIQP). GMAC® Research Report RR-14-01

    ERIC Educational Resources Information Center

    Han, Kyung T.; Rudner, Lawrence M.

    2014-01-01

    This study uses mixed integer quadratic programming (MIQP) to construct multiple highly equivalent item pools simultaneously, and compares the results from mixed integer programming (MIP). Three different MIP/MIQP models were implemented and evaluated using real CAT item pool data with 23 different content areas and a goal of equal information…

  5. Final construction quality assurance report for the Y-12 Industrial Landfill V, Area 2, Oak Ridge, Tennessee

    SciTech Connect

    Bessom, W.H.

    1996-11-01

    Lockheed Martin Energy Systems (LMES) has finished construction of Area 2 of the Y-12 Plant Industrial Landfill (ILF-V), classified as a Class 2 Landfill. This final Construction Quality Assurance (CQA) Report provides documentation that Area 2 was constructed in substantial compliance with the Tennessee Department of Environment and Conservation (TDEC) approved design, as indicated and specified in the permit drawings, approved changes, and specifications. This report applies specifically to the Area 2 excavation, compacted clay soil liner, geomembrane liner, granular leachate collection layer, protective soil cover, and the leachate collection system. An ``As-Built`` survey was performed and is included. The drawings provide horizontal and vertical information for Area 2, the anchor trench, the leachate collection pipe, the temporary access road, and cross-sections of Area 2. This report provides documentation of the following items: the excavation activities of Area 2; the maximum recompacted coefficient of hydraulic conductivity or permeability of the soil is less than 1 {times} 10{sup {minus}7} centimeters per second (cm/sec); the total thickness of the compacted clay soil liner equals a minimum of 2 feet; a 40 mil impermeable geomembrane (polypropylene) flexible membrane liner (FML) and 16 oz. geotextile fabric was placed in direct contact with the compacted clay soil liner; a 12 inch granular leachate collection layer was installed and covered with a 8 oz. geotextile separation fabric; the installation of the leachate collection piping; and the two foot protective clay soil cover.

  6. LSST summit facility construction progress report: reacting to design refinements and field conditions

    NASA Astrophysics Data System (ADS)

    Barr, Jeffrey D.; Gressler, William; Sebag, Jacques; Seriche, Jaime; Serrano, Eduardo

    2016-07-01

    The civil work, site infrastructure and buildings for the summit facility of the Large Synoptic Survey Telescope (LSST) are among the first major elements that need to be designed, bid and constructed to support the subsequent integration of the dome, telescope, optics, camera and supporting systems. As the contracts for those other major subsystems now move forward under the management of the LSST Telescope and Site (T and S) team, there has been inevitable and beneficial evolution in their designs, which has resulted in significant modifications to the facility and infrastructure. The earliest design requirements for the LSST summit facility were first documented in 2005, its contracted full design was initiated in 2010, and construction began in January, 2015. During that entire development period, and extending now roughly halfway through construction, there continue to be necessary modifications to the facility design resulting from the refinement of interfaces to other major elements of the LSST project and now, during construction, due to unanticipated field conditions. Changes from evolving interfaces have principally involved the telescope mount, the dome and mirror handling/coating facilities which have included significant variations in mass, dimensions, heat loads and anchorage conditions. Modifications related to field conditions have included specifying and testing alternative methods of excavation and contending with the lack of competent rock substrate where it was predicted to be. While these and other necessary changes are somewhat specific to the LSST project and site, they also exemplify inherent challenges related to the typical timeline for the design and construction of astronomical observatory support facilities relative to the overall development of the project.

  7. Choline Acetyltransferase Activity in Striatum of Neonatal Rats Increased by Nerve Growth Factor

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Rutkowski, J. Lynn; Tennekoon, Gihan I.; Buchanan, Karen; Johnston, Michael V.

    1985-07-01

    Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

  8. Final audit report of remedial action construction at the UMTRA Project Mexican Hat, Utah -- Monument Valley, Arizona, sites

    SciTech Connect

    1995-10-01

    The final audit report for remedial action at the Mexican Hat, Utah, Monument Valley, Arizona, Uranium Mill Tailings Remedial Action (UMTRA) Project sites consists of a summary of the radiological surveillances/audits, quality assurance (QA) in-process surveillances, and QA remedial action close-out inspections performed by the US Department of Energy (DOE) and the Technical Assistance Contractor (TAC); on-site construction reviews (OSCR) performed by the US Nuclear Regulatory Commission (NRC); and a surveillance performed by the Navajo Nation. This report refers to remedial action activities performed at the Mexican Hat, Utah--Monument Valley, Arizona, Uranium Mill Tailings Remedial Action (UMTRA) Project sites.

  9. 7 CFR 3015.85 - Outlay report and request for reimbursement for construction programs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... (Continued) OFFICE OF THE CHIEF FINANCIAL OFFICER, DEPARTMENT OF AGRICULTURE UNIFORM FEDERAL ASSISTANCE REGULATIONS Financial Reporting Requirements § 3015.85 Outlay report and request for reimbursement for... § 3015.84. (3) The awarding agency may substitute the Financial Status Report specified in § 3015.82...

  10. Prototypical Rod Construction Demonstration Project. Phase 3, Final report: Volume 1, Cold checkout test report, Book 3

    SciTech Connect

    Not Available

    1993-05-01

    The objective of Phase 3 of the Prototypical Rod consolidation Demonstration Project (PRCDP) was to procure, fabricate, assemble, and test the Prototypical Rod consolidation System as described in the NUS Phase 2 Final Design Report. This effort required providing the materials, components, and fabricated parts which makes up all of the system equipment. In addition, it included the assembly, installation, and setup of this equipment at the Cold Test Facility. During the Phase 3 effort the system was tested on a component, subsystem, and system level. This volume 1, discusses the PRCDP Phase 3 Test Program that was conducted by the HALLIBURTON NUS Environmental Corporation under contract AC07-86ID12651 with the United States Department of Energy. This document, Volume 1, Book 3 discusses the following topics: Downender Test Results and Analysis Report; NFBC Canister Upender Test Results and Analysis Report; Fuel Assembly Handling Fixture Test Results and Analysis Report; and Fuel Canister Upender Test Results and Analysis Report.

  11. Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

    SciTech Connect

    Thoden, James B.; Reinhardt, Laurie A.; Cook, Paul D.; Menden, Patrick; Cleland, W.W.; Holden, Hazel M.

    2012-09-17

    N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed {beta}-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 {angstrom} resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel {beta}-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed {beta}-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

  12. Choline acetyltransferase: further studies on the reverse reaction

    SciTech Connect

    Hsu, L.L.; Chao, L.P.

    1982-01-01

    In order to further characterize the reaction mechanism of brain ChAc in its purified form, we have investigated the reverse reaction of ChAc in terms of pH optimum, salt effects, and substrate kinetics using a radiochemical assay. We directly measured the reaction product acetylcoenzyme A which was separated from the substrate ACh by a cation exchange column. Dowex 50W-X8 (Na+ form). The reverse reaction of ChAc was linear with incubation time up to 40 minutes, and with enzyme protein concentration up to 5 micrograms. It had a pH optimum at 7.0. At 0.22 M the monovalent chloride and bromide salts activated the reverse ChAc activity by 23-47% but the fluoride and iodide salts inhibited the reverse enzyme activity by 10-30%. Kinetic studies in the absence of salt showed that KACh was 0.62 +/- 0.06 mM, KCoA . SH was 12.68 +/- 1.21 microM, and Vmax was 11.6 +/- 1.0 nmol AcCoA/mg protein/min. These data are in disagreement with the values reported on partially purified ChAc from bovine brain by Glover and Potter (1971) and Hersh (1980). This indicates that further investigations are necessary to clarify or resolve these differences.

  13. Up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) expression is a part of proliferative but not anabolic response of mouse kidney.

    PubMed

    Dudkowska, Magdalena; Stachurska, Agnieszka; Grzelakowska-Sztabert, Barbara; Manteuffel-Cymborowska, Małgorzata

    2002-01-01

    A differential expression pattern of spermidine/spermine N(1)-acetyltransferase (SSAT), the enzyme critical to proper homeostasis of cellular polyamines, is reported in mouse kidney undergoing hyperplasia and hypertrophy. We have shown that SSAT activity and SSAT mRNA are significantly induced by antifolate CB 3717 and folate that evoke a drug-injury-dependent hyperplasia. In contrast, SSAT activity is down-regulated in the testosterone-induced hypertrophic kidney, while SSAT mRNA is positively controlled by this androgen. Catecholamine depletion evoked by reserpine drastically decreases the folate-induced activity of S-adenosylmethionine decarboxylase (AdoMetDC), which limits polyamine biosynthesis, but has no effect on SSAT activity augmented by CB 3717. Our results document that the increased SSAT expression solely accompanies the proliferative response of mouse kidney, and suggest the importance of post-transcriptional regulation to the control of SSAT activity in both hyperplastic and hypertrophic experimental models.

  14. No association between apolipoprotein E or N‐Acetyltransferase 2 gene polymorphisms and age‐related hearing loss

    PubMed Central

    Dawes, Piers; Platt, Hazel; Horan, Michael; Ollier, William; Munro, Kevin; Pendleton, Neil

    2014-01-01

    Objectives/Hypothesis Age‐related hearing loss has a genetic component, but there have been limited genetic studies in this field. Both N‐acetyltransferase 2 and apolipoprotein E genes have previously been associated. However, these studies have either used small sample sizes, examined a limited number of polymorphisms, or have produced conflicting results. Here we use a haplotype tagging approach to determine association with age‐related hearing loss and investigate epistasis between these two genes. Study Design Candidate gene association study of a continuous phenotype. Methods We investigated haplotype tagging single nucleotide polymorphisms in the N‐acetyltransferase 2 gene and the presence/absence of the apolipoprotein E ε4 allele for association with age‐related hearing loss in a cohort of 265 Caucasian elderly volunteers from Greater Manchester, United Kingdom. Hearing phenotypes were generated using principal component analysis of the hearing threshold levels for the better ear (severity, slope, and concavity). Genotype data for the N‐acetyltransferase 2 gene was obtained from existing genome‐wide association study data from the Illumina 610‐Quadv1 chip. Apolipoprotein E genotyping was performed using Sequenom technology. Linear regression analysis was performed using Plink and Stata software. Results No significant associations (P value, > 0.05) were observed between the N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms and any hearing factor. No significant association was observed for epistasis analysis of apolipoprotein E ε4 and the N‐acetyltransferase 2 single nucleotide polymorphism rs1799930 (NAT2*6A). Conclusion We found no evidence to support that either N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms are associated with age‐related hearing loss in a cohort of 265 elderly volunteers. Level of Evidence N/A. Laryngoscope, 125:E33–E38, 2015 PMID:25155015

  15. Construction of Primary Healthcare Centers Reported Essentially Complete, but Operational Issues Remain

    DTIC Science & Technology

    2009-04-29

    in Iraq. In addition, the task orders provided for the delivery and installation of medical and dental equipment at the PHCs. Two years later...stated that neither the construction nor the materials met expected standards and the facilities did not make a good presentation of a U.S.-funded and...Type C, about 2,126 square meters). In addition, the three task orders provided for the delivery and installation of medical and dental equipment at

  16. A Revised Simplex Method for Test Construction Problems. Research Report 90-5.

    ERIC Educational Resources Information Center

    Adema, Jos J.

    Linear programming models with 0-1 variables are useful for the construction of tests from an item bank. Most solution strategies for these models start with solving the relaxed 0-1 linear programming model, allowing the 0-1 variables to take on values between 0 and 1. Then, a 0-1 solution is found by just rounding, optimal rounding, or a…

  17. Phytoremediation of explosives contaminated groundwater in constructed wetlands: 2. Flow through study. Draft report

    SciTech Connect

    DBehrends, L.L.; Sikora, F.J.; Phillips, W.D.; Baily, E.; McDonald, C.

    1996-02-01

    This study evaluates the utility of constructed wetlands for remediating explosives contaminated groundwaters using bench scale flow-through type reactors. Specifially the study examines: the degradation of TNT, TNB, RDX, and HMX in contaminated waters in plant lagoons and gravel-based wetlands. The study also provides design recommendations for the wetland demonstration project to be located at the Milan Army Ammunition Plant (MAAP), in Tennessee.

  18. Demonstration of constructed wetlands for treatment of municipal wastewaters, monitoring report for the period, March 1988--October 1989

    SciTech Connect

    Choate, K.D.; Watson, J.T.; Steiner, G.R.

    1990-08-01

    To evaluate the constructed wetland technology, the Tennessee Valley Authority (TVA) implemented a municipal wastewater demonstration project in western Kentucky. Using combined city, State, and TVA appropriated funds, three constructed wetland systems were built at Benton, Hardin, and Pembroke, Kentucky. Demonstration objectives include evaluating relative advantages and disadvantages of these types of systems; determining permit compliance ability; developing, evaluating, and improving basic design and operation criteria; evaluating cost effectiveness; and transferring technology to users and regulators. A demonstration monitoring project was implemented with a partnership of funds from the Environmental Protection Agency (EPA) Region IV, other EPA funds through the National Small Flows Clearinghouse (NSFC), and TVA appropriations. TVA is managing the project in cooperation with an interagency team consisting of EPA, Kentucky Division of Water and NSFC. This report, which supersedes the first monitoring report (Choate, et. al., 1989) of these demonstration projects, describes each constructed wetland system, its status, and summarizes monitoring data and plans for each system. 5 refs., 30 figs., 26 tabs.

  19. Educational Reform Implementation: A Co-Constructed Process. Research Report 5.

    ERIC Educational Resources Information Center

    Datnow, Amanda; Hubbard, Lea; Mehan, Hugh

    This research report argues for viewing the complex, often messy process of school reform implementation as a "conditional matrix" coupled with qualitative research. As illustration, two studies (of six reform efforts in one county and of implementation of an untracking program in Kentucky) are reported. Preliminary analysis reveals that…

  20. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

    PubMed Central

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

  1. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  2. Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-l-carnitine

    PubMed Central

    Chase, J. F. A.; Tubbs, P. K.

    1970-01-01

    Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[14C]bromoacetyl-l-carnitine yields N-3-carboxy[14C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue. PMID:5461620

  3. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

    PubMed Central

    Casini, A.; Vaccaro, R.; D'Este, L.; Sakaue, Y.; Bellier, J.P.; Kimura, H.; Renda, T.G.

    2012-01-01

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes. PMID:23027350

  4. An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Rather, P N; Parojcic, M M; Paradise, M R

    1997-08-01

    The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.

  5. An acetyltransferase-independent function of Eso1 regulates centromere cohesion

    PubMed Central

    Lin, Su-Jiun; Tapia-Alveal, Claudia; Jabado, Omar J.; Germain, Doris; O’Connell, Matthew J.

    2016-01-01

    Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe. We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes. PMID:27798241

  6. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  7. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization*

    PubMed Central

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-01-01

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism. PMID:26251518

  8. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization.

    PubMed

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-09-18

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

  9. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

    PubMed

    Casini, A; Vaccaro, R; D'Este, L; Sakaue, Y; Bellier, J P; Kimura, H; Renda, T G

    2012-07-19

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

  10. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase

    PubMed Central

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-01-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

  11. Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

    PubMed

    Green, Keith D; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C; Tsodikov, Oleg V; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

  12. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  13. The Chromatin Regulator BRPF3 Preferentially Activates the HBO1 Acetyltransferase but Is Dispensable for Mouse Development and Survival*

    PubMed Central

    Yan, Kezhi; You, Linya; Degerny, Cindy; Ghorbani, Mohammad; Liu, Xin; Chen, Lulu; Li, Lin; Miao, Dengshun; Yang, Xiang-Jiao

    2016-01-01

    To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of β-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2. PMID:26677226

  14. Rocky Mountain Arsenal Northwest Boundary Containment/Treatment System Construction Report. Text, Drawings, Photos

    DTIC Science & Technology

    1986-03-01

    inch by lb-inch DuPont "Tovex" water gel with a 25 millisecond delay between holes . The two blasts used patterns of 2-inch thinwall PVC cased holes in...14 January 1983. I Key ’WORD$ (Conch0 on prevr o4do Hf Ŕeeeev OWid Ieneir fty &o e*k mA.r) Ground Water Hydrologyi’.) Soil-Bentonite Cutoff Barrier1...system was constructed to contain and treat ground water which has been polluted with organic contaminants produced at the arsenal. The system consists

  15. Construction Foundation Report, Missouri River, Fort Peck Lake, Montana. Volume 1. Text and Photos.

    DTIC Science & Technology

    1983-01-01

    projecting ends of the back walls in the 20-foot blocks, with these wales braced to poured niches in the completed concrete. All shale surfaces were sealed...steel. Many vertical braces between the tiers of steel and additional struts were required in order to hold the trenches until concrete could be poured...Preparation 52 4.6 Safety Precautions 54 4.7 Permanent Foundation Anchors 54 Chapter 5 - Special Foundation Construction’ 55 5.1 Main Embankment Cutoff Wall 56

  16. Life-management strategies of selection, optimization, and compensation: measurement by self-report and construct validity.

    PubMed

    Freund, Alexandra M; Baltes, Paul B

    2002-04-01

    The authors examined the usefulness of a self-report measure for elective selection, loss-based selection. optimization, and compensation (SOC) as strategies of life management. The expected 4-factor solution was obtained in 2 independent samples (N = 218, 14-87 years; N = 181, 18-89 years) exhibiting high retest stability across 4 weeks (r(tt) = .74-82). As expected, middle-aged adults showed higher endorsement of SOC than younger and older adults. Moreover, SOC showed meaningful convergent and divergent associations to other psychological constructs (e.g., thinking styles, NEO) and evinced positive correlations with measures of well-being which were maintained after other personality and motivational constructs were controlled for. Initial evidence on behavioral associations involving SOC obtained in other studies is summarized.

  17. Native American Children and Their Reports of Hope: Construct Validation of the Children’s Hope Scale

    PubMed Central

    Boles, Richard E.; Roberts, Michael C.; Winston, Lauren

    2014-01-01

    Child reports of hope continue to be utilized as predictors of positive adjustment; however, the utilization of the hope construct has not been assessed within the culturally diverse Native American child group. The present study investigated the applicability of the Hope theory among 96 Native American children in the Midwest. Measures included the Children’s Hope Scale and a Hope Interview. Native American children in the current sample appear to conceptualize hope as a way to reach goals as did the children in the normative sample. Results from the factor analysis demonstrate that the factor structure found in the current study was similar to the factor structure found in the standardization sample. Because of the similar Hope theory conceptualization and factor structure, interventions focused on the positive psychology construct of hope may be applicable within a Native American child population. PMID:26622164

  18. Energy efficient residential new construction: market transformation. Spectral selective glass. Final project report

    SciTech Connect

    Hammon, Robert

    2000-12-18

    This final report describes the following tasks associated with this project: cost and availability of spectrally selective glass (SSG); window labeling problem and field verification of glass; availability of SSG replacement glass and tempered glass; HVAC load reduction due to spectrally selective glass; and comsumer appreciation of spectrally selective glass. Also included in the report are four attachments: builder and HVAC subcontractor presentation, sample advertisements, spectrally selective glass demonstration model, and invitation to SCE Glass mini trade-show.

  19. Aloe-emodin inhibited N-acetylation and DNA adduct of 2-aminofluorene and arylamine N-acetyltransferase gene expression in mouse leukemia L 1210 cells.

    PubMed

    Chung, Jing-Gung; Li, Yu-Ching; Lee, Yi-Min; Lin, Jing-Pin; Cheng, Kwork-Chui; Chang, Weng-Cheng

    2003-09-01

    N-Acetyltransferases (NATs) plays an important role in the first step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylatoion phenotypes, which are recognized to affect cancer risk related to environmental exposure. Aloe-emodin has been shown to exit anticancer activity. The purpose of this study is to examine whether or not aloe-emodin could affect arylamine N-acetyltransferase (NAT) activity and gene expression (NAT mRNA) and DNA-2-aminofluorene (DNA-AF) adduct formation in mouse leukemia cells (L 1210). By using high performance liquid chromatography, N-acetylation and non-N-acetylation of AF were determined and quantitated. By using reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, NAT mRNA was determined and quantitated. Aloe-emodin displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by aloe-emodin for up to 24h. Using standard steady-state kinetic analysis, it was demonstrated that aloe-emodin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA-AF adduct formation in mouse leukemia cells were inhibited by aloe-emodin. The NAT1 mRNA in mouse leukemia cells were also inhibited by aloe-emodin. This report is the first demonstration which showed aloe-emodin affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and DNA-AF on adduct formation.

  20. Spatial memory consolidation is associated with induction of several lysine-acetyltransferase (histone acetyltransferase) expression levels and H2B/H4 acetylation-dependent transcriptional events in the rat hippocampus.

    PubMed

    Bousiges, Olivier; Vasconcelos, Anne Pereira de; Neidl, Romain; Cosquer, Brigitte; Herbeaux, Karine; Panteleeva, Irina; Loeffler, Jean-Philippe; Cassel, Jean-Christophe; Boutillier, Anne-Laurence

    2010-12-01

    Numerous genetic studies have shown that the CREB-binding protein (CBP) is an essential component of long-term memory formation, through its histone acetyltransferase (HAT) function. E1A-binding protein p300 and p300/CBP-associated factor (PCAF) have also recently been involved in memory formation. By contrast, only a few studies have reported on acetylation modifications during memory formation, and it remains unclear as to how the system is regulated during this dynamic phase. We investigated acetylation-dependent events and the expression profiles of these HATs during a hippocampus-dependent task taxing spatial reference memory in the Morris water maze. We found a specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated. This increase correlated with the degree of specific acetylated histones enrichment on some memory/plasticity-related gene promoters. Overall, a global increase in HAT activity was measured during this memory consolidation phase, together with a global increase of CBP, p300, and PCAF expression. Interestingly, these regulations were altered in a model of hippocampal denervation disrupting spatial memory consolidation, making it impossible for the hippocampus to recruit the CBP pathway (CBP regulation and acetylated-H2B-dependent transcription). CBP has long been thought to be present in limited concentrations in the cells. These results show, for the first time, that CBP, p300, and PCAF are dynamically modulated during the establishment of a spatial memory and are likely to contribute to the induction of a specific epigenetic tagging of the genome for hippocampus-dependent (spatial) memory consolidation. These findings suggest the use of HAT-activating molecules in new therapeutic strategies of pathological aging, Alzheimer's disease, and other neurodegenerative disorders.

  1. Mitigation for the Construction and Operation of Libby Dam, 2000 Annual Report.

    SciTech Connect

    Hoffman, Greg; Marotz, Brian L.; Dunnigan, James

    2002-09-01

    ''Mitigation for the Construction and Operation of Libby Dam'' is part of the Northwest Power Planning Council's resident fish and wildlife program. The program was mandated by the Northwest Planning Act of 1980, and is responsible for mitigating for damages to fish and wildlife caused by hydroelectric development in the Columbia River Basin. The objective of Phase I of the project (1983 through 1987) was to maintain or enhance the Libby Reservoir fishery by quantifying seasonal water levels and developing ecologically sound operational guidelines. The objective of Phase II of the project (1988 through 1996) was to determine the biological effects of reservoir operations combined with biotic changes associated with an aging reservoir. The objectives of Phase III of the project (1996 through present) are to implement habitat enhancement measures to mitigate for dam effects, to provide data for implementation of operational strategies that benefit resident fish, monitor reservoir and river conditions, and monitor mitigation projects for effectiveness.

  2. Concrete sandwich construction for energy conservation. Final report, October 1975-September 1978

    SciTech Connect

    Keeton, J.R.

    1980-03-01

    An abbreviated research study on use of shrinkage-compensating expansive concrete in sandwich-type wall and roof panels containing insulation at mid-thickness is described. The use of expansive concrete is shown to be a technically viable concept for eliminating shrinkage cracking, thus preventing moisture penetration which can reduce insulation effectiveness, cause deterioration of the insulating material, and accelerate steel corrosion. Embeddable resistance strain gages proved to be reliable for measuring expansion and subsequent shrinkage of the experimental panels. As a result of this study, a comprehensive research program is proposed for experimental verification of design and field control measures that will permit the use of shrinkage-compensating cement mortars in sandwich panel construction.

  3. Earthquake resistant construction of electric transmission and telecommunication facilities serving the Federal government report

    SciTech Connect

    Yokel, F.Y.

    1990-02-01

    The vulnerability of electrical transmission and telecommunication facilities to damage in past earthquakes, as well as available standards and technologies to protect these facilities against earthquake damage are reviewed. An overview is presented of measures taken by various Federal agencies to protect electrical transmission and telecommunication facilities against earthquake hazards. It is concluded that while most new facilities which are owned and operated by Federal agencies are presently designed to provide some, though not necessarily adequate, earthquake resistance, there generally is no effort to retrofit existing facilities. No evidence was found of requirements to protect electrical transmission and communication facilities which have major contractual obligations to serve the Federal Government and only limited seismic design requirements are stipulated for electrical transmission systems constructed with Federal funding.

  4. Constructed wetlands for municipal solid waste landfill leachate treatment. Final report

    SciTech Connect

    Peverly, J.; Sanford, W.E.; Steenhuis, T.S.

    1993-11-01

    In 1989, the US Geological Survey and Cornell University, in cooperation with the New York State Energy Research and Development Authority and the Tompkins County Solid Waste Department, began a three-year study at a municipal solid-waste landfill near Ithaca, New York, to test the effectiveness of leachate treatment with constructed wetlands and to examine the associated treatment processes. Specific objectives of the study were to examine: treatment efficiency as function of substrate composition and grain size, degree of plant growth, and seasonal changes in evapotranspiration rates and microbial activity; effects of leachate and plant growth on the hydraulic characteristics of the substrate; and chemical, biological, and physical processes by which nutrients, metals, and organic compounds are removed from leachate as it flows through the substrate. A parallel study at a municipal solid-waste landfill near Fenton, New York was conducted by researchers at Cornell University, Ithaca College, and Hawk Engineering (Trautmann and others, 1989). Results are described.

  5. E-ELT M4 adaptive unit final design and construction: a progress report

    NASA Astrophysics Data System (ADS)

    Biasi, Roberto; Manetti, Mauro; Andrighettoni, Mario; Angerer, Gerald; Pescoller, Dietrich; Patauner, Christian; Gallieni, Daniele; Tintori, Matteo; Mantegazza, Marco; Fumi, Pierluigi; Lazzarini, Paolo; Briguglio, Runa; Xompero, Marco; Pariani, Giorgio; Riccardi, Armando; Vernet, Elise; Pettazzi, Lorenzo; Lilley, Paul; Cayrel, Marc

    2016-07-01

    The E-ELT M4 adaptive unit is a fundamental part of the E-ELT: it provides the facility level adaptive optics correction that compensates the wavefront distortion induced by atmospheric turbulence and partially corrects the structural deformations caused by wind. The unit is based on the contactless, voice-coil technology already successfully deployed on several large adaptive mirrors, like the LBT, Magellan and VLT adaptive secondary mirrors. It features a 2.4m diameter flat mirror, controlled by 5316 actuators and divided in six segments. The reference structure is monolithic and the cophasing between the segments is guaranteed by the contactless embedded metrology. The mirror correction commands are usually transferred as modal amplitudes, that are checked by the M4 controller through a smart real-time algorithm that is capable to handle saturation effects. A large hexapod provides the fine positioning of the unit, while a rotational mechanism allows switching between the two Nasmyth foci. The unit has entered the final design and construction phase in July 2015, after an advanced preliminary design. The final design review is planned for fall 2017; thereafter, the unit will enter the construction and test phase. Acceptance in Europe after full optical calibration is planned for 2022, while the delivery to Cerro Armazones will occur in 2023. Even if the fundamental concept has remained unchanged with respect to the other contactless large deformable mirrors, the specific requirements of the E-ELT unit posed new design challenges that required very peculiar solutions. Therefore, a significant part of the design phase has been focused on the validation of the new aspects, based on analysis, numerical simulations and experimental tests. Several experimental tests have been executed on the Demonstration Prototype, which is the 222 actuators prototype developed in the frame of the advanced preliminary design. We present the main project phases, the current design

  6. Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p.

    PubMed

    Ter Veld, Frank; Wolff, Daniel; Schorsch, Christoph; Köhler, Tim; Boles, Eckhard; Poetsch, Ansgar

    2013-10-01

    Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.

  7. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified...

  8. Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-spermidine/spermine acetyltransferase (SSAT)

    SciTech Connect

    Forouhar,F.; Lee, I.; Vujcic, J.; Vujcic, S.; Shen, J.; Vorobiev, S.; Xiao, R.; Acton, T.; Montelione, G.; et al.

    2005-01-01

    Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates (SSATs). We have determined the crystal structure of PaiA in complex with CoA at 1.9 Angstrom resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility since purified PaiA possesses N1-acetyltransferase activity towards polyamine substrates including spermidine and spermine. Further, conditional over-expression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

  9. Arylamine N-acetyltransferases: a structural perspective. Comments regarding the BJP paper by Zhou et al., 2013

    PubMed Central

    Xu, Ximing; Kubiak, Xavier; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2014-01-01

    This letter is a comment on Zhou et al. (2013). Arylamine N-acetyltransferases: a structural perspective. Br J Pharmacol 169: 748–760. To view this article visit http://dx.doi.org/10.1111/bph.12182 PMID:24328723

  10. Comparative genomic, phylogenetic, and functional investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the...

  11. Automatic Dictionary Construction; Part II of Scientific Report No. ISR-18, Information Storage and Retrieval...

    ERIC Educational Resources Information Center

    Cornell Univ., Ithaca, NY. Dept. of Computer Science.

    Part Two of the eighteenth report on Salton's Magical Automatic Retriever of Texts (SMART) project is composed of three papers: The first: "The Effect of Common Words and Synonyms on Retrieval Performance" by D. Bergmark discloses that removal of common words from the query and document vectors significantly increases precision and that…

  12. Solar Energy School Heating Augmentation Experiment. Design, Construction and Initial Operation. A Report.

    ERIC Educational Resources Information Center

    InterTechnology Corp., Warrenton, VA.

    This report describes an experimental solar heating system, complete with thermal storage and controls, that has met all the heating requirements of five detached classrooms of the Fauquier High School in Warrenton, Virginia. The objectives of the experiment were to (1) demonstrate that solar energy can be used to provide a substantial part of the…

  13. Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2016-09-01

    The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 μm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event.

  14. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveal Substrate Specificity of Protein Acetyltransferases*

    PubMed Central

    Crosby, Heidi A.; Pelletier, Dale A.; Hurst, Gregory B.; Escalante-Semerena, Jorge C.

    2012-01-01

    N-Lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously. PMID:22416131

  15. Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis.

    PubMed

    Ambrosone, Christine B; Kropp, Silke; Yang, Jun; Yao, Song; Shields, Peter G; Chang-Claude, Jenny

    2008-01-01

    Approximately 10 years ago, it was noted that smoking increased risk of breast cancer among women with N-acetyltransferase 2 (NAT2) slow acetylation genotypes. This report was followed by a number of studies to address this question. We pooled data from 10 existing studies and also conducted a meta-analysis of 13 studies published from 1996 to October 2006 that were conducted among women, were published in English, and had adequate information on smoking and NAT2 genotyping. Raw data were requested from authors. Unconditional logistic regression was done for pooled analysis, and random effect models was done for meta-analysis. Study heterogeneity was assessed, and sensitivity tests were done when subgroups were excluded from the analysis. In the pooled analysis, there was a significant interaction between smoking, NAT2 genotype, and risk of breast cancer [pack-years (continuous variable, P(interaction) = 0.03)], with higher pack-years significantly associated with an increased risk of breast cancer among women with NAT2 slow genotypes (pooled analysis relative risk, 1.49; 95% confidence interval, 1.08-2.04). These findings were supported by the meta-analysis including all studies; pack-years were significantly associated with risk among slow acetylators in a dose-dependent fashion (meta-analysis relative risk, 1.44; 95% confidence interval, 1.23-1.68 for > or =20 pack-years versus never smokers), but not among rapid acetylators. Similar relationships were noted for smoking status (ever, never) and duration of smoking. Our results show that cigarette smoking is associated with an increase in breast cancer risk among women with NAT2 slow acetylation genotypes. Because slow NAT2 genotypes are present in 50% to 60% of Caucasian populations, smoking is likely to play an important role in breast cancer etiology.

  16. N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

    PubMed Central

    Van Damme, Petra; Lasa, Marta; Polevoda, Bogdan; Gazquez, Cristina; Elosegui-Artola, Alberto; Kim, Duk Soo; De Juan-Pardo, Elena; Demeyer, Kimberly; Hole, Kristine; Larrea, Esther; Timmerman, Evy; Prieto, Jesus; Arnesen, Thomas; Sherman, Fred; Gevaert, Kris; Aldabe, Rafael

    2012-01-01

    Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human. PMID:22814378

  17. Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis.

    PubMed

    Damron, F Heath; McKenney, Elizabeth S; Barbier, Mariette; Liechti, George W; Schweizer, Herbert P; Goldberg, Joanna B

    2013-07-01

    We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

  18. The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

    PubMed

    Ding, Bao-Jian; Lager, Ida; Bansal, Sunil; Durrett, Timothy P; Stymne, Sten; Löfstedt, Christer

    2016-04-01

    Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

  19. Mitigation for the Construction and Operation of Libby Dam, 2004-2005 Annual Report.

    SciTech Connect

    Dunnigan, James; DeShazer, Jay; Garrow, Larry

    2005-06-01

    ''Mitigation for the Construction and Operation of Libby Dam'' is part of the Northwest Power and Conservation Council's (NPCC) resident fish and wildlife program. The program was mandated by the Northwest Planning Act of 1980, and is responsible for mitigating damages to fish and wildlife caused by hydroelectric development in the Columbia River Basin. The objective of Phase I of the project (1983 through 1987) was to maintain or enhance the Libby Reservoir fishery by quantifying seasonal water levels and developing ecologically sound operational guidelines. The objective of Phase II of the project (1988 through 1996) was to determine the biological effects of reservoir operations combined with biotic changes associated with an aging reservoir. The objectives of Phase III of the project (1996 through present) are to implement habitat enhancement measures to mitigate for dam effects, to provide data for implementation of operational strategies that benefit resident fish, monitor reservoir and river conditions, and monitor mitigation projects for effectiveness. This project completes urgent and high priority mitigation actions as directed by the Kootenai Subbasin Plan. Montana Fish, Wildlife & Parks (MFWP) uses a combination of techniques to collect physical and biological data within the Kootenai River Basin. These data serve several purposes including: the development and refinement of models used in management of water resources and operation of Libby Dam; investigations into the limiting factors of native fish populations, gathering basic life history information, tracking trends in endangered and threatened species, and the assessment of restoration or management activities designed to restore native fishes and their habitats.

  20. An examination of the construct and predictive validity of the self-reported speeding behavior model.

    PubMed

    Jovanović, Dragan; Šraml, Matjaž; Matović, Boško; Mićić, Spasoje

    2017-02-01

    The present study deals with the problem of speeding behavior on rural roads. The purpose of the paper is to examine the construct validity and the internal consistency and reliability of a questionnaire that measures the determinants of speeding behavior. In addition, it aimed to test the predictive validity of a modified theoretical framework of a theory of planned behavior (TPB) in relation to speeding behavior. A total of 546 car drivers from five local communities in the Republic of Srpska successfully completed the questionnaire after reading the scenario. The principal component analysis revealed seven components interpreted as: personal norm, perceived behavioral control, affective attitude toward speeding, subjective norm, habit, descriptive norm, and cognitive attitude toward speeding. A speeding behavior model was developed by structural equation modeling. Personal norm, subjective norm, and affective attitudes were shown to be important variables within the modified TPB in understanding speeding behavior. Overall, the present findings provide significant support for the concept of the modified theoretical framework of TPB in relation to speeding behavior on rural roads. Implications for a speeding behavior model and interventions are discussed.

  1. Tazimina hydroelectric project, Iliamna, Alaska. Final technical and construction cost report

    SciTech Connect

    1998-08-01

    The Iliamna-Newhalen-Nondalton Electric Cooperative (INNEC) provides electrical power to three communities of the same names. These communities are located near the north shore of Iliamna Lake in south-central Alaska approximately 175 miles southwest of Anchorage. A hydroelectric project was constructed for these communities, starting in the spring of 1996 and ending in the spring of 1998. The project site is on the Tazimina River about 12 miles northeast of Iliamna Lake. The taximina River flows west from the Aleutian Range. The project site is at Tazimina Falls about 9 miles upstream of the confluence of the Tazimina River and the Newhalen River. The project has an installed capacity of 824 kilowatts (kW) and is expandable to 1.5 megawatts (MW). The project is run-of-the-river (no storage) and uses the approximately 100 feet of natural head provided by the falls. The project features include a channel control sill, intake structure, penstock, underground powerhouse, tailrace, surface control building, buried transmission line and communication cable, and access road.

  2. Tazimina Hydroelectric Project, Iliamna, Alaska Final Technical and Construction Cost Report

    SciTech Connect

    HDR Alaska, Inc.

    1998-11-01

    The Iliamna-Newhalen-Nondalton Electric Cooperative (INNEC) provides electrical power to three communities of the same names. These communities are located near the north shore of Iliamna Lake in south-central Alaska approximately 175 miles southwest of Anchorage. These communities have a combined population of approximately 600 residents. There is no direct road connection from these villages to larger population centers. Electric power has been generated by INNEC since 1983 using diesel generators located in the community of Newhalen. Fuel for these generators was transported up the Kvichak River, an important salmon river, and across Iliamna Lake. In dry years the river is low and fuel is flown into Iliamna and then trucked five miles into Newhalen. The cost, difficult logistics and potential spill hazard of this fuel was a primary reason for development of hydroelectric power in this area. A hydroelectric project was constructed for these communities, starting in the spring of 1996 and ending in the spring of 1998. The project site is at Tazimina Falls about 9 miles upstream of the confluence of the Tazimina River and the Newhalen River. The project has an installed capacity of 824 kilowatts (kW) and is expandable to 1.5 megawatts (MW). The project is run-of-the-river (no storage) and uses the approximately 100 feet of natural head provided by the falls. The project features include a channel control sill, intake structure, penstock, underground powerhouse, tailrace, surface control building, buried transmission line and communication cable, and access road.

  3. Ohio Coal Testing and Development Facility - Construction and operation. Final report

    SciTech Connect

    Ferris, D.D.

    1996-03-01

    On June 14, 1987, the Ohio Coal Development Office (OCDO) executed a grant agreement with ICF Kaiser Engineers (ICF Kaiser) for the planning and design (Phase I) of a Demonstration Advanced Technology Coal Preparation Facility. Subsequently, on December 1, 1990, OCDO executed a grant agreement with the American Electric Power Service Corporation (AEPSC) through its subsidiary, the Ohio Power Company, for the final design and construction (Phase II), testing and operation (Phase III), and marketing and future operation (Phase IV) of the facility. These phases were subcontracted to ICF Kaiser. AEPSC co-sponsored the project and donated a site at the Central Ohio Coal Company`s Unionville Coal Preparation Plant for locating the test plant. Central Ohio Coal supplied coal handling services, waste-product disposal, and water. The Ohio Power Company provided project oversight, electric power, and the test coals. The test results from the operation of the 30 tph advanced coal cleaning plant demonstrated that combining conventional physical coal cleaning with emerging advanced physical coal cleaning technologies was a cost-effective method to reduce sulfur emissions of Ohio coals. The following is a summary of the key findings of this project.

  4. Investigation of the ways in which patients' reports of their satisfaction with healthcare are constructed.

    PubMed

    Edwards, Carol; Staniszweska, Sophie; Crichton, Nicola

    2004-03-01

    A characteristic feature of patient satisfaction research is the consistently high level of satisfaction recorded. More reliable and relevant inquiry tools are constantly being developed, but underlying psychological and social pressures that could promote such a consistent and undiscriminating response have been little investigated. Williams et al. (1998) explored the phenomenon and concluded that, by considering issues of duty and culpability, patients could make allowances for poor care, and avoid evaluating it negatively. Their study was in community mental health. This study follows up their work within elective orthopaedic surgery, and investigates the pressures promoting such apparent transformation of opinion. Using a longitudinal design, and in-depth qualitative interviews, the patient's process of reflection was explored. Three psycho-social pressures were identified that appear to work together to make the transformation of opinion almost the default process. They are: the relative dependency of patients within the healthcare system; their need to maintain constructive working relationships with those providing their care; and their general preference for holding a positive outlook. It is suggested that, while it is the patient's prerogative to re-interpret the quality of their care positively, it is not the prerogative of the inquirer to accept this re-interpretation as representative of the patient's experience. Methods of inquiry are needed which access something of patients' development of opinion, and thereby something of their initial, often more negative, untransformed responses to their healthcare experiences.

  5. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    SciTech Connect

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  6. Borehole summary report for five ground-water monitoring wells constructed in the 1100 Area

    SciTech Connect

    Bryce, R.W.; Goodwin, S.M.

    1989-05-01

    This report contains the data collected during the installation and initial sampling of five ground-water monitoring wells between the 1100 Area and Richland City water supply wells. The five wells were installed to provide for early detection of contaminants and to provide data that may be used in making decisions on the management of the North Richland Well Field and recharge basins. 2 refs., 1 fig.

  7. A Guide to Soil Reports and Differing Site Conditions for Construction Professionals

    DTIC Science & Technology

    1990-12-01

    Foundation Engineering Handbook, Van Nostrand Reinhold Company, New York, NY., Page 3. 87 Ibid., Pages 4 /5. 88 Ibid., Page 6. 89 C.R.I. Clayton, N.E...ASCE, 110( 4 ). Dowding, C.H., 1978, Site Characterization & Exploration, American Society of Civil Engineers, New York, NY. Gaskins, "Changed Conditions...listed and discussed_. gommon problems in soil reports which have led to court cases.are also discussed. , 4 n aj iii TABLE OF CONTENTS LIST OF

  8. Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.

    PubMed

    Lambrechts, Dennis; Roeffaers, Maarten; Kerckhofs, Greet; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

    2014-09-01

    By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.

  9. Mitigation for the Construction and Operation of Libby Dam, 2003-2004 Annual Report.

    SciTech Connect

    Dunnigan, James; DeShazer, Jay; Garrow, Larry

    2004-06-01

    ''Mitigation for the Construction and Operation of Libby Dam'' is part of the Northwest Power and Conservation Council's (NPCC) resident fish and wildlife program. The program was mandated by the Northwest Planning Act of 1980, and is responsible for mitigating for damages to fish and wildlife caused by hydroelectric development in the Columbia River Basin. The objective of Phase I of the project (1983 through 1987) was to maintain or enhance the Libby Reservoir fishery by quantifying seasonal water levels and developing ecologically sound operational guidelines. The objective of Phase II of the project (1988 through 1996) was to determine the biological effects of reservoir operations combined with biotic changes associated with an aging reservoir. The objectives of Phase III of the project (1996 through present) are to implement habitat enhancement measures to mitigate for dam effects, to provide data for implementation of operational strategies that benefit resident fish, monitor reservoir and river conditions, and monitor mitigation projects for effectiveness. This project completes urgent and high priority mitigation actions as directed by the Kootenai Subbasin Plan. Montana FWP uses a combination of diverse techniques to collect a variety of physical and biological data within the Kootenai River Basin. These data serve several purposes including: the development and refinement of models used in management of water resources and operation of Libby Dam; investigations into the limiting factors of native fish populations, gathering basic life history information, tracking trends in endangered, threatened species, and the assessment of restoration or management activities intended to restore native fishes and their habitats.

  10. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

    SciTech Connect

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie

    2011-09-06

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  11. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  12. Comparison of protein acetyltransferase action of CRTAase with the prototypes of HAT.

    PubMed

    Ponnan, Prija; Kumar, Ajit; Singh, Prabhjot; Gupta, Prachi; Joshi, Rini; Gaspari, Marco; Saso, Luciano; Prasad, Ashok K; Rastogi, Ramesh C; Parmar, Virinder S; Raj, Hanumantharao G

    2014-01-01

    Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

  13. Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis.

    PubMed

    Karukurichi, Kannan R; Wang, Ling; Uzasci, Lerna; Manlandro, Cara Marie; Wang, Qing; Cole, Philip A

    2010-02-03

    The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulates HAT activity. Here we have developed a strategy to introduce acetyl-Lys at up to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA substrate analogue and that HAT activity correlates positively with degree of acetylation. This study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.

  14. Circadian clock controlling arylalkylamine N-acetyltransferase-like activity in the cricket (Gryllus bimaculatus) egg.

    PubMed

    Itoh, M T; Sumi, Y

    1998-07-13

    When cricket (Gryllus bimaculatus) eggs were incubated under a 12-h light/12-h dark (LD) cycle for 6 days after oviposition at 24-26 degrees C and thereafter transferred to constant darkness (DD), arylalkylamine N-acetyltransferase (NAT)-like activity fluctuated in a circadian manner, peaking during the subjective dark period, and the rhythmic activity persisted during the 3rd day of incubation in DD. When the eggs were transferred from LD to a lighting regime in which the light and dark periods were reversed, the rhythm of NAT-like activity continued to oscillate in phase with the light/dark cycle. These data demonstrate that the cricket egg (probably the embryo) contains a circadian clock controlling NAT-like activity, and that the circadian clock entrains to environmental light/dark cycles.

  15. Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

    SciTech Connect

    Creighton, J.A.; Rudeen, P.K.

    1988-01-01

    The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.

  16. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  17. Involvement of Arabidopsis histone acetyltransferase HAC family genes in the ethylene signaling pathway.

    PubMed

    Li, Chao; Xu, Jiang; Li, Jian; Li, Qingyun; Yang, Hongchun

    2014-02-01

    Epigenetic modifications play a fundamental role in regulating chromatin dynamics and gene expression. The level of histone acetylation is controlled by two functionally antagonistic enzymes, namely histone acetyltransferase (HAT) and histone deacetylase (HDAC). CREB-binding protein (CBP)/p300 proteins, a subfamily of highly conserved HATs, are involved in various physiological events including proliferation, differentiation and apoptosis. In this work, we study the poorly known function of their homologous genes, the HAC genes, in Arabidopsis. We found that hac1-involved mutants displayed pleiotropic phenotypes, in particular hypersensitivity to ethylene both in the dark and in the light. We also found that the transcriptional levels of ethylene-responsive genes are significantly higher in the hac1hac5 double mutant than in wild-type plants. Moreover, an ethylene synthesis inhibitor cannot release the triple responses of hac mutants. These results suggest that HACs are involved in the ethylene signaling pathway.

  18. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    SciTech Connect

    Coppel, R.L.; McNeilage, L.J.; Surh, C.D.; Van De Water, J.; Spithill, T.W.; Whittingham, S.; Gershwin, M.E. )

    1988-10-01

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a M{sub r} 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

  19. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

    PubMed Central

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J.; Martinho, Rui Gonçalo

    2016-01-01

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes. PMID:26861501

  20. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases

    SciTech Connect

    Wu,H.; Dombrovsky, L.; Tempel, W.; Martin, F.; Loppnau, P.; Goodfellow, G.; Grant, D.; Plotnikov, A.

    2007-01-01

    The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

  1. The histone acetyltransferase MOF activates hypothalamic polysialylation to prevent diet-induced obesity in mice

    PubMed Central

    Brenachot, Xavier; Rigault, Caroline; Nédélec, Emmanuelle; Laderrière, Amélie; Khanam, Tasneem; Gouazé, Alexandra; Chaudy, Sylvie; Lemoine, Aleth; Datiche, Frédérique; Gascuel, Jean; Pénicaud, Luc; Benani, Alexandre

    2014-01-01

    Overfeeding causes rapid synaptic remodeling in hypothalamus feeding circuits. Polysialylation of cell surface molecules is a key step in this neuronal rewiring and allows normalization of food intake. Here we examined the role of hypothalamic polysialylation in the long-term maintenance of body weight, and deciphered the molecular sequence underlying its nutritional regulation. We found that upon high fat diet (HFD), reduced hypothalamic polysialylation exacerbated the diet-induced obese phenotype in mice. Upon HFD, the histone acetyltransferase MOF was rapidly recruited on the St8sia4 polysialyltransferase-encoding gene. Mof silencing in the mediobasal hypothalamus of adult mice prevented activation of the St8sia4 gene transcription, reduced polysialylation, altered the acute homeostatic feeding response to HFD and increased the body weight gain. These findings indicate that impaired hypothalamic polysialylation contribute to the development of obesity, and establish a role for MOF in the brain control of energy balance. PMID:25161885

  2. Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

    PubMed

    Muoio, Deborah M; Noland, Robert C; Kovalik, Jean-Paul; Seiler, Sarah E; Davies, Michael N; DeBalsi, Karen L; Ilkayeva, Olga R; Stevens, Robert D; Kheterpal, Indu; Zhang, Jingying; Covington, Jeffrey D; Bajpeyi, Sudip; Ravussin, Eric; Kraus, William; Koves, Timothy R; Mynatt, Randall L

    2012-05-02

    The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

  3. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis.

    PubMed

    Garzan, Atefeh; Willby, Melisa J; Green, Keith D; Gajadeera, Chathurada S; Hou, Caixia; Tsodikov, Oleg V; Posey, James E; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  4. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB.

    PubMed

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R; Tsodikov, Oleg V; Garneau-Tsodikova, Sylvie

    2011-06-14

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  5. Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

    PubMed

    Willby, Melisa J; Green, Keith D; Gajadeera, Chathurada S; Hou, Caixia; Tsodikov, Oleg V; Posey, James E; Garneau-Tsodikova, Sylvie

    2016-06-17

    A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

  6. Erosion Control of Scour during Construction. Report 7. Current--A Wave-Induced Current Model.

    DTIC Science & Technology

    1984-09-01

    Engineers Waewybxermn tto VP0 Box 631, Vicksburg, Mississippi 39180-0631 84 11 21 065 i ~ . ,o. .. ~--.’" Destroy this report when oolonger needed. Do not...to 1.99 $PAR) 2 EY Eddy viscosity c (ft /sec) y RAD A weighting factor (between zero and one) for radiation stress gradient terms NTIMEB Number of...time-steps over which radiation terms are to be built up ADVI A weighting factor (between zero and one) for advection terms FRCI A weighting factor

  7. The place of choline acetyltransferase activity measurement in the "cholinergic hypothesis" of neurodegenerative diseases.

    PubMed

    Contestabile, Antonio; Ciani, Elisabetta; Contestabile, Andrea

    2008-02-01

    The so-called "cholinergic hypothesis" assumes that degenerative dysfunction of the cholinergic system originating in the basal forebrain and innervating several cortical regions and the hippocampus, is related to memory impairment and neurodegeneration found in several forms of dementia and in brain aging. Biochemical methods measuring the activity of the key enzyme for acetylcholine synthesis, choline acetyltransferase, have been used for many years as a reliable marker of the integrity or the damage of the cholinergic pathways. Stereologic counting of the basal forebrain cholinergic cell bodies, has been additionally used to assess neurodegenerative changes of the forebrain cholinergic system. While initially believed to mark relatively early stages of disease, cholinergic dysfunction is at present considered to occur in advanced dementia of Alzheimer's type, while its involvement in mild and prodromal stages of the disease has been questioned. The issue is relevant to better understand the neuropathological basis of the diseases, but it is also of primary importance for therapy. During the last few years, indeed, cholinergic replacement therapies, mainly based on the use of acetylcholinesterase inhibitors to increase synaptic availability of acetylcholine, have been exploited on the assumption that they could ameliorate the progression of the dementia from its initial stages. In the present paper, we review data from human studies, as well as from animal models of Alzheimer's and Down's diseases, focusing on different ways to evaluate cholinergic dysfunction, also in relation to the time point at which these dysfunctions can be demonstrated, and on some discrepancy arising from the use of different methodological approaches. The reviewed literature, as well as some recent data from our laboratories on a mouse model of Down's syndrome, stress the importance of performing biochemical evaluation of choline acetyltransferase activity to assess cholinergic

  8. Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides

    SciTech Connect

    Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan

    2009-08-14

    Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the 'in vivo' characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

  9. Molecular Determinants of the N-Terminal Acetyltransferase Naa60 Anchoring to the Golgi Membrane.

    PubMed

    Aksnes, Henriette; Goris, Marianne; Strømland, Øyvind; Drazic, Adrian; Waheed, Qaiser; Reuter, Nathalie; Arnesen, Thomas

    2017-02-14

    Nα-acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) since it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N-termini of transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to study secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C-terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C-terminus is unstructured in solution and only folds into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 towards membranes containing the phosphatidylinositol PI4P, thus possibly explaining the primary residency of Naa60 at the PI4P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

  10. Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa

    PubMed Central

    Norris, Adrianne L; Serpersu, Engin H

    2013-01-01

    Aminoglycoside-modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase-IIa (AAC-IIa) and its comparison to two highly promiscuous AGMEs, the AG N3-acetyltransferase-IIIb (AAC-IIIb) and the AG phosphotransferase(3′)-IIIa (APH). Despite having similar antibiotic selectivities, AAC-IIIb and APH catalyze different reactions and share no homology to one another. AAC-IIa and AAC-IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC-IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well-defined conformations when AGs are bound. Contrary to this AAC-IIIb maintains a well-defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme. PMID:23640799

  11. Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation

    PubMed Central

    Sun, Wu-Sheng; Chun, Ju-Lan; Do, Jeong-Tae; Kim, Dong-Hwan; Ahn, Jin-Seop; Kim, Min-Kyu

    2016-01-01

    Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population. PMID:27999597

  12. Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation.

    PubMed

    Sun, Wu-Sheng; Chun, Ju-Lan; Do, Jeong-Tae; Kim, Dong-Hwan; Ahn, Jin-Seop; Kim, Min-Kyu; Hwang, In-Sul; Kwon, Dae-Jin; Hwang, Seong-Soo; Lee, Jeong-Woong

    2016-01-01

    Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5' upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.

  13. A new self-report inventory of dyslexia for students: criterion and construct validity.

    PubMed

    Tamboer, Peter; Vorst, Harrie C M

    2015-02-01

    The validity of a Dutch self-report inventory of dyslexia was ascertained in two samples of students. Six biographical questions, 20 general language statements and 56 specific language statements were based on dyslexia as a multi-dimensional deficit. Dyslexia and non-dyslexia were assessed with two criteria: identification with test results (Sample 1) and classification using biographical information (both samples). Using discriminant analyses, these criteria were predicted with various groups of statements. All together, 11 discriminant functions were used to estimate classification accuracy of the inventory. In Sample 1, 15 statements predicted the test criterion with classification accuracy of 98%, and 18 statements predicted the biographical criterion with classification accuracy of 97%. In Sample 2, 16 statements predicted the biographical criterion with classification accuracy of 94%. Estimations of positive and negative predictive value were 89% and 99%. Items of various discriminant functions were factor analysed to find characteristic difficulties of students with dyslexia, resulting in a five-factor structure in Sample 1 and a four-factor structure in Sample 2. Answer bias was investigated with measures of internal consistency reliability. Less than 20 self-report items are sufficient to accurately classify students with and without dyslexia. This supports the usefulness of self-assessment of dyslexia as a valid alternative to diagnostic test batteries.

  14. Re-Use of Clean Coal Technology By-Products in the Construction of Low Permeability Liners. Final report

    SciTech Connect

    Wolfe, William E.; Butalia, Tarunjit S.; Walker, Harold; Mitsch, William

    2005-07-15

    This final project report presents the results of a research program conducted at The Ohio State University from January 3, 2000 to June 30, 2005 to investigate the long-term use of stabilized flue gas desulfurization (FGD) materials in the construction of low permeability liners for ponds and wetlands. The objective of the research program was to establish long-term field-verified time-dependent relationships for the performance of liners constructed from stabilized FGD byproducts generated in Ohio. The project objective was accomplished with a coordinated program of testing and analyzing small-scale laboratory specimens under controlled conditions, mediumscale wetland experiments, and monitoring of a full-scale FGD-lined pond facility. Although the specific uses directly addressed by this report include liners for surface impoundments, the results presented in this study are also useful in other applications especially in the design of daily covers and liners for landfills, seepage cutoff walls and trenches, and for nutrient retention and pollution mitigation wetlands. The small-scale laboratory tests and monitoring of the full-scale FGD lined facility (capacity of one million gallons) shows that stabilized FGD materials can be used as low permeability liners in the construction of water and manure holding ponds. Actual long-term permeability coefficients in the range of 10-7 cm/sec (3 x 10-9 ft/sec) can be obtained in the field by compacting lime and fly ash enriched stabilized FGD materials. Leachate from the FGD material meets Ohio’s non-toxic criteria for coal combustion by-products, and for most potential contaminants the national primary and secondary drinking water standards are also met. The low permeability non-toxic FGD material investigated in this study poses very minimal risks, if any, for groundwater contamination. The FGD wetland experiments indicated no significant differences in phosphorus retention between the clay and FGD

  15. Laboratory Investigations Relevant to Cassini VIMS Reports of Coherent Constructive Interference in Saturn's Rings

    NASA Astrophysics Data System (ADS)

    Nelson, Robert

    The Cassini spacecraft has flown between the sun and Saturn on several occasions during its orbital tour creating circumstances in which the zero phase point passed through the rings. The Visual and Infrared Mapping Spectrometer (VIMS) recorded spectral image cubes (0.4 to 5.2 microns that showed the opposition effect (OE) at zero phase. The OE is a spike in the intensity of reflected light observed near zero phase when it is displayed as a function of phase angle. This is the first time the OE has been resolved for small areas on the rings. Laboratory investigations of angular scattering properties of particulate materials show that the OE arises from two distinct processes, shadow hiding (SHOE) and coherent backscattering (CBOE). The SHOE process causes an OE by the elimination of shadows cast by regolith grains upon one another as phase angle decreases. The CBOE process causes an OE by constructive interference between photons traveling in opposite directions along the same path within the medium. SHOE is expected to dominate the contribution to the OE in absorbing media where multiple scattering of photons is not significant. CBOE is expected to dominate the contribution to the OE in highly reflective media with much multiple scattering. We have made spectral dimension scans of the VIMS images that traverse the zero phase point. We selected narrow spectral bands that represent a variety of wavelengths and reflectance levels. In this way, phase curves of the ringlet were obtained for each band. We have compared these data to data we acquired in the laboratory using the JPL long arm goniometer where we measured the phase curve of particulate materials that simulate the surface of Saturn's ring particles. We argue here that the OE is due to coherent backscattering because: 1) The theoretical CBOE function fit to the data is excellent. 2) The OE width is extremely narrow 3) The angular width of the peak increases with wavelength. CBOE theory also predicts that the

  16. Design, construction and testing of a DC bioeffects test enclosure for small animals. Final report

    SciTech Connect

    Frazier, M J; Preache, M M

    1980-11-01

    This final report describes both the engineering development of a DC bioeffects test enclosure for small laboratory animals, and the biological protocol for the use of such enclosures in the testing of animals to determine possible biological effects of the environment associated with HVDC transmission lines. The test enclosure which has been designed is a modular unit, which will house up to eight rat-sized animals in individual compartments. Multiple test enclosures can be used to test larger numbers of animals. A prototype test enclosure has been fabricated and tested to characterize its electrical performance characteristics. The test enclosure provides a simulation of the dominant environment associated with HVDC transmission lines; namely, a static electric field and an ion current density. A biological experimental design has been developed for assessing the effects of the dominant components of the HVDC transmission line environment.

  17. Final audit report of remedial action construction at the UMTRA Project Ambrosia Lake, New Mexico, site

    SciTech Connect

    1995-09-01

    The final audit report for remedial action at the Ambrosia Lake, New Mexico, Uranium Mill Tailings Remedial Action (UMTRA) Project site consists of a summary of the radiological surveillances/audits, quality assurance (QA) in-process surveillances, and a QA final closeout inspection performed by the US Department of Energy (DOE) and the Technical Assistance Contractor (TAC). One radiological surveillance and three radiological audits were performed at the Ambrosia Lake site. The surveillance was performed on 12--16 April 1993 (DOE, 1993d). The audits were performed on 26--29 July 1993 (DOE, 1993b); 21--23 March 1994 (DOE, 1994d); and 1--2 August 1994 (DOE, 1994d). The surveillance and audits resulted in 47 observations. Twelve of the observations raised DOE concerns that were resolved on site or through subsequent corrective action. All outstanding issues were satisfactorily closed out on 28 December 1994. The radiological surveillance and audits are discussed in this report. A total of seven QA in-process surveillances were performed at the Ambrosia Lake UMTRA site are discussed. The DOE/TAC Ambrosia Lake final remedial action close-out inspection was conducted on 26 July 1995 (DOE, 1995a). To summarize, a total of 155 observations were noted during DOE/TAC audit and surveillance activities. Follow-up to responses required from the RAC for the DOE/TAC surveillance and audit observations indicated that all issues related to the Ambrosia Lake site were resolved and closed to the satisfaction of the DOE.

  18. Libby Mitigation Program, 2007 Annual Progress Report: Mitigation for the Construction and Operation of Libby Dam.

    SciTech Connect

    Dunnigan, James; DeShazer, J.; Garrow, L.

    2009-05-26

    basin streams and lakes. 'Mitigation for the Construction and Operation of Libby Dam' is part of the Northwest Power and Conservation Council's (NPCC) resident fish and wildlife program. The program was mandated by the Northwest Planning Act of 1980, and is responsible for mitigating damages to fish and wildlife caused by hydroelectric development in the Columbia River Basin. The objective of Phase I of the project (1983 through 1987) was to maintain or enhance the Libby Reservoir fishery by quantifying seasonal water levels and developing ecologically sound operational guidelines. The objective of Phase II of the project (1988 through 1996) was to determine the biological effects of reservoir operations combined with biotic changes associated with an aging reservoir. The objectives of Phase III of the project (1996 through present) are to implement habitat enhancement measures to mitigate for dam effects, to provide data for implementation of operational strategies that benefit resident fish, monitor reservoir and river conditions, and monitor mitigation projects for effectiveness. This project completes urgent and high priority mitigation actions as directed by the Kootenai Subbasin Plan.

  19. Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed

    Macinga, D R; Parojcic, M M; Rather, P N

    1995-06-01

    The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498).

  20. Final audit report of remedial action construction at the UMTRA Project, Grand Junction, Colorado, processing site

    SciTech Connect

    1995-02-01

    This final audit report (FAR) for remedial action at the Grand Junction, Colorado, Uranium Mill Tailings Remedial Action (UMTRA) Project processing site consists of a summary of the radiological surveillances/ audits, the quality assurance (QA) in-process surveillances, and the QA final close-out inspection performed by the US Department of Energy (DOE) and Technical Assistance Contractor (TAC). The FAR also summarizes other surveillances performed by the US Nuclear Regulatory Commission (NRC). To summarize, a total of one finding and 127 observations were noted during DOE/TAC audit and surveillance activities. The NRC noted general site-related observations during the OSCRs. Follow-up to responses required from MK-Ferguson for the DOE/TAC finding and observations indicated that all issues related to the Grand Junction processing site were resolved and closed out to the DOE`s satisfaction. The NRC OSCRs resulted in no issues related to the Grand Junction processing site requiring a response from MK-Ferguson.

  1. Measuring teacher self-report on classroom practices: Construct validity and reliability of the Classroom Strategies Scale-Teacher Form.

    PubMed

    Reddy, Linda A; Dudek, Christopher M; Fabiano, Gregory A; Peters, Stephanie

    2015-12-01

    This article presents information about the construct validity and reliability of a new teacher self-report measure of classroom instructional and behavioral practices (the Classroom Strategies Scales-Teacher Form; CSS-T). The theoretical underpinnings and empirical basis for the instructional and behavioral management scales are presented. Information is provided about the construct validity, internal consistency, test-retest reliability, and freedom from item-bias of the scales. Given previous investigations with the CSS Observer Form, it was hypothesized that internal consistency would be adequate and that confirmatory factor analyses (CFA) of CSS-T data from 293 classrooms would offer empirical support for the CSS-T's Total, Composite and subscales, and yield a similar factor structure to that of the CSS Observer Form. Goodness-of-fit indices of χ2/df, Root Mean Square Error of Approximation, Goodness of Fit Index, and Adjusted Goodness of Fit Index suggested satisfactory fit of proposed CFA models whereas the Comparative Fit Index did not. Internal consistency estimates of .93 and .94 were obtained for the Instructional Strategies and Behavioral Strategies Total scales respectively. Adequate test-retest reliability was found for instructional and behavioral total scales (r = .79, r = .84, percent agreement 93% and 93%). The CSS-T evidences freedom from item bias on important teacher demographics (age, educational degree, and years of teaching experience). Implications of results are discussed.

  2. Evaluation and utilization of Illinois FBC residues for construction materials. Technical report, September 1, 1991--November 30, 1991

    SciTech Connect

    Ghafoori, N.

    1991-12-31

    The overall objective of this program is to investigate the extent to which fluidized bed combustion (FBC) by-products can be properly utilized as the viable construction materials. This investigation focuses primarily on the properties of residues derived from fluidized combustion burning of Illinois high-sulfur coal. The research plan calls for evaluation of physico-chemical and engineering characteristics of the FBC-based cement and non-cement mixes. The results of this study will be used to compare the physical and mechanical properties of the FBC-based mixtures with those of conventional mixes. The suitability of using FBC residues as a filler or binder aggregate for construction applications such as structural concrete members, precast building products, and as base or surface course for gravity dams and pavements in the form of conventional and roller compacted materials will then be evaluated. During this reporting period, the literature survey, preparation of raw materials, and chemical analyses were completed. Some of the physical properties and preconditioning studies were determined and efforts are in progress to complete these tasks.

  3. Construction of a bacterium to convert cellulose to ethanol. Final report

    SciTech Connect

    Bellamy, W.D.

    1984-03-01

    In the strains of thermophilic actinomycetes examined, cellobiase (CBase) and Beta-glucosidase (BGSase) were determined to be separate enzymes. Both enzymes are induced by cellulose, cellobiose and lactose. A number of strains do not utilize lactose. Lactose does not induce endocellulase (CMCase) in any of the strains examined. In all the strains examined, the CBase and BGSase were far more heat labile than the extracellular CMCase. The 50% survival time at 60/sup 0/C is as follows: CMCase, 24 hrs; CBase, 10 to 11 hrs; BGSase, 2 to 5 hrs. The BGSase and CBase of Clostridium thermocellum are more heat resistant with 50% survival times: BGSase, 14 hrs; CBase, 41 hrs. Whey permeate is an adequate substrate for a number of strains if supplemented with 0.1% yeast extract or biotin and thiamine. It is speculated that whey permeate could be used for commercial production of CBase and BGSase. All attempts to produce a thermophilic bacillus that was ethanol-tolerant and produced high yields of ethanol by induced mutation using ultraviolet radiation and N-methyl-N'-nitrosogunidine as mutagens were unsuccessful. No evidence was observed that the Acetyl-S-CoA metabolic pathway was deleted or suppressed. Some of the mutants appeared to have decreased yields of lactic acid. A satisfactory screening procedure for selection of high ethanol producing colonies was not found. The screening for low acid production was tedious and time consuming. Because of the failure to find or produce a thermophile with high yields of ethanol, and because all previous work as reported in the literature also yielded poor results, it may be impossible to produce an ethanol-tolerant high yielding thermophilic microorganism. The essential proteins may be unstable at greater than 7% ethanol at 55 to 66/sup 0/C. 48 references, 6 figures, 16 tables.

  4. Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

    PubMed Central

    Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

    2011-01-01

    A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

  5. Regulation of KAT6 Acetyltransferases and Their Roles in Cell Cycle Progression, Stem Cell Maintenance, and Human Disease

    PubMed Central

    Huang, Fu

    2016-01-01

    The lysine acetyltransferase 6 (KAT6) histone acetyltransferase (HAT) complexes are highly conserved from yeast to higher organisms. They acetylate histone H3 and other nonhistone substrates and are involved in cell cycle regulation and stem cell maintenance. In addition, the human KAT6 HATs are recurrently mutated in leukemia and solid tumors. Therefore, it is important to understand the mechanisms underlying the regulation of KAT6 HATs and their roles in cell cycle progression. In this minireview, we summarize the identification and analysis of the KAT6 complexes and discuss the regulatory mechanisms governing their enzymatic activities and substrate specificities. We further focus on the roles of KAT6 HATs in regulating cell proliferation and stem cell maintenance and review recent insights that aid in understanding their involvement in human diseases. PMID:27185879

  6. Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

    PubMed

    Weiser, J; Buriánková, K; Kalachová, L; Branny, P; Pernodet, J L

    2006-01-01

    A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

  7. Constructed Wetlands for Treatment of Organic and Engineered Nanomaterial Contaminants of Emerging Concerns (WaterRF Report 4334)

    EPA Science Inventory

    The goal of this project was to determine hydraulic and carbon loading rates for constructed wetlands required for achieving different levels of organic and nanomaterial contaminants of emerging concern (CECs) removal in constructed wetlands. Specific research objectives included...

  8. A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation

    PubMed Central

    Duan, Cheng-Guo; Wang, Xingang; Xie, Shaojun; Pan, Li; Miki, Daisuke; Tang, Kai; Hsu, Chuan-Chih; Lei, Mingguang; Zhong, Yingli; Hou, Yueh-Ju; Wang, Zhijuan; Zhang, Zhengjing; Mangrauthia, Satendra K; Xu, Huawei; Zhang, Heng; Dilkes, Brian; Tao, W Andy; Zhu, Jian-Kang

    2017-01-01

    Transposons are generally kept silent by epigenetic mechanisms including DNA methylation. Here, we identified a pair of Harbinger transposon-derived proteins (HDPs), HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were co-domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDM1, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. HDP2 and the methyl-DNA-binding protein MBD7 share a large set of common genomic binding sites, indicating that they jointly determine the target specificity of the histone acetyltransferase complex. Thus, our data revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing. PMID:27934869

  9. Generation IV Nuclear Energy Systems Construction Cost Reductions through the Use of Virtual Environments - Task 5 Report: Generation IV Reactor Virtual Mockup Proof-of-Principle Study

    SciTech Connect

    Timothy Shaw; Anthony Baratta; Vaughn Whisker

    2005-02-28

    Task 5 report is part of a 3 year DOE NERI-sponsored effort evaluating immersive virtual reality (CAVE) technology for design review, construction planning, and maintenance planning and training for next generation nuclear power plants. Program covers development of full-scale virtual mockups generated from 3D CAD data presented in a CAVE visualization facility. Created a virtual mockup of PBMR reactor cavity and discussed applications of virtual mockup technology to improve Gen IV design review, construction planning, and maintenance planning.

  10. 42 CFR 137.351 - Is a Self-Governance Tribe required to submit construction project progress and financial reports...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., DEPARTMENT OF HEALTH AND HUMAN SERVICES TRIBAL SELF-GOVERNANCE Construction Roles of Self-Governance Tribe in Establishing and Implementing Construction Project Agreements § 137.351 Is a Self-Governance Tribe required to... Self-Governance Tribe must provide the Secretary with construction project progress and...

  11. Evaluation of building design, construction, and performance for the control of radon in florida houses. Evaluation of radon resistant construction techniques in eight new houses. Final report, January 1993-April 1994

    SciTech Connect

    Hintenlang, D.E.; Shanker, A.; Najafi, F.T.; Roessler, E.C.

    1995-07-01

    The report gives results of a study of eight houses throughout their construction in North Central Florida. Each house was built in compliance with the proposed radon resistant construction standard being developed by the Florida Department of Community Affairs. Each house was monitored for at least 6 days after construction, operating in three different heating, ventilation, and air-conditioning (HVAC) system configurations. Continuous measurements of indoor radon concentrations, house ventilation rates, across-slab differential pressures, and interzone differential pressures provided time-resolved radon entry rates and a performance index for passive radon barriers. Radon entry rates were found to be relatively constant throughout the measurement periods and for the different house operating conditions, implying that the passive radon barrier eliminates most convective entry.

  12. A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii.

    PubMed

    Galopin, Sébastien; Cattoir, Vincent; Leclercq, Roland

    2009-06-01

    The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat(Bcl), coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat(Bcl) gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat-specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat(Bcl) was chromosomally located in all four resistant B. clausii strains.

  13. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    SciTech Connect

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  14. [Evaluation of a caffeine test for determining the phenotype of N-acetyltransferase].

    PubMed

    Gascon, M P; Leemann, T; Dayer, P

    1987-12-05

    Xenobiotic acetylation by N-acetyltransferase is genetically controlled. This polymorphism governs the intestinal and liver metabolism of numerous amines. The use of caffeine, a ubiquitous and nontoxic amine, has been proposed as a probe for phenotyping. The aim of the present study is to evaluate this test and to identify the metabolite of caffeine used as substrate by the polymorphic enzyme. - A cup of coffee, tea or Coca-Cola is administered to fasting subjects. The molar ratio of two metabolites of caffeine (AFMU and 1X) is determined on a spot urine sample 4-6 hours later by means of a UV liquid chromatographic assay. In a reference population (n = 63), the distribution of molar ratios is trimodal with frequencies of 0.14, 0.35 and 0.51. These results correlate with those obtained by the classic isoniazid test. However, in vitro experiments in human liver subcellular fractions did not lead to the identification of a xanthine as the precursor of the acetylated metabolite.

  15. Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs).

    PubMed

    Gorsuch, Stephen; Bavetsias, Vassilios; Rowlands, Martin G; Aherne, G Wynne; Workman, Paul; Jarman, Michael; McDonald, Edward

    2009-01-15

    High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

  16. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair

    PubMed Central

    Bassi, C; Li, Y-T; Khu, K; Mateo, F; Baniasadi, P S; Elia, A; Mason, J; Stambolic, V; Pujana, M A; Mak, T W; Gorrini, C

    2016-01-01

    The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation. PMID:26915295

  17. Polyamine regulation of heat-shock-induced spermidine N1-acetyltransferase activity.

    PubMed Central

    Fuller, D J; Carper, S W; Clay, L; Chen, J R; Gerner, E W

    1990-01-01

    The enzyme spermidine/spermine N1-acetyltransferase (N1-SAT) is rapidly induced by heat shock in CHO and A549 cells, with activity declining by 24 h. Depletion of intracellular polyamines by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, blocks this induction. Re-addition of putrescine to these cultures restores the response to heat shock, with a concomitant increase in intracellular N1-acetylspermidine. Diaminopropane is more than twice as effective as the naturally occurring diamine putrescine, suggesting that the propylamine moiety of spermidine is involved in the regulation of N1-SAT induction. Inhibitor studies indicate transcriptional activation and that the enzyme has an apparent half-life of 30-60 min. A second heat shock rapidly inhibits induced N1-SAT activity, which decays with a half-life of 2-3 min. Despite its induction by heat, N1-SAT is not a stable enzyme, suggesting that the activity observed is not due to a modification of an existing peptide, but is due to a transcriptional event, which may justify the inclusion of this enzyme in the family of heat-shock proteins. Images Fig. 2. PMID:2111132

  18. Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

    PubMed Central

    2015-01-01

    Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

  19. Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii.

    PubMed

    Payie, K G; Rather, P N; Clarke, A J

    1995-08-01

    A collection of Providencia stuartii mutants which either underexpress or overexpress aac(2')-Ia, the chromosomal gene coding for gentamicin 2'-N-acetyltransferase (EC 2.3.1.59), have been characterized phenotypically as possessing either lower or higher levels of peptidoglycan O acetylation, respectively, than the wild type. These mutants were subjected to both negative-staining and thin-section electron microscopy. P. stuartii PR100, with 42% O acetylation of peptidoglycan compared with 52% O acetylation in the wild type, appeared as irregular rods. In direct contrast, P. stuartii strains PR50.LM3 and PR51, with increased levels of peptidoglycan O acetylation (65 and 63%, respectively), appeared as coccobacilli and chain formers, respectively. Membrane blebbing was also observed with the chain-forming strain PR51. Thin sectioning of this mutant indicated that it was capable of proper constriction and separation. P. stuartii PM1, when grown to mid-exponential phase, did not have altered peptidoglycan O-acetylation levels, and cellular morphology remained similar to that of wild-type strains. However, continued growth into stationary phase resulted in a 15% increase in peptidoglycan O acetylation concomitant with a change of some cells from a rod-shaped to a coccobacillus-shaped morphology. The fact that these apparent morphological changes were directly related to levels of O acetylation support the view that this modification plays a role in the maintenance of peptidoglycan structure, presumably through the control of autolytic activity.

  20. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene

    PubMed Central

    Knowles, Joshua W.; Xie, Weijia; Zhang, Zhongyang; Chennemsetty, Indumathi; Assimes, Themistocles L.; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O.; Carcamo-Orive, Ivan; Morris, Andrew P.; Chen, Yii-Der I.; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M.; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J.; Tsao, Philip S.; Schadt, Eric E.; Rotter, Jerome I.; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A.; Groop, Leif; Cordell, Heather J.; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M.; Weedon, Michael N.; Walker, Mark; Quertermous, Thomas

    2015-01-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 “A” allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity. PMID:25798622

  1. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    SciTech Connect

    Fungjou Lu; Youngshin Chen . Dept. of Biochemistry); Tienshang Huang . Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  2. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    PubMed Central

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  3. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

    PubMed

    Liu, Haijing; Ling, Yun; Gong, Yilei; Sun, Ying; Hou, Lin; Zhang, Bo

    2007-06-01

    NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

  4. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  5. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

  6. Choline acetyltransferase in the hippocampus is associated with learning strategy preference in adult male rats.

    PubMed

    Hawley, Wayne R; Witty, Christine F; Daniel, Jill M; Dohanich, Gary P

    2015-08-01

    One principle of the multiple memory systems hypothesis posits that the hippocampus-based and striatum-based memory systems compete for control over learning. Consistent with this notion, previous research indicates that the cholinergic system of the hippocampus plays a role in modulating the preference for a hippocampus-based place learning strategy over a striatum-based stimulus--response learning strategy. Interestingly, in the hippocampus, greater activity and higher protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, are associated with better performance on hippocampus-based learning and memory tasks. With this in mind, the primary aim of the current study was to determine if higher levels of ChAT and the high-affinity choline uptake transporter (CHT) in the hippocampus were associated with a preference for a hippocampus-based place learning strategy on a task that also could be solved by relying on a striatum-based stimulus--response learning strategy. Results confirmed that levels of ChAT in the dorsal region of the hippocampus were associated with a preference for a place learning strategy on a water maze task that could also be solved by adopting a stimulus-response learning strategy. Consistent with previous studies, the current results support the hypothesis that the cholinergic system of the hippocampus plays a role in balancing competition between memory systems that modulate learning strategy preference.

  7. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  8. The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

    PubMed Central

    Ehrenhofer-Murray, A. E.; Rivier, D. H.; Rine, J.

    1997-01-01

    Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed. PMID:9093847

  9. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

    PubMed

    Zaunbrecher, M Analise; Sikes, R David; Metchock, Beverly; Shinnick, Thomas M; Posey, James E

    2009-11-24

    The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)

  10. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury

    PubMed Central

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

  11. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey

    PubMed Central

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L.; Shi, Qiong

    2015-01-01

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2. PMID:26729109

  12. Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

    PubMed

    Park, Sangkyu; Byeon, Yeong; Lee, Hyoung Yool; Kim, Young-Soon; Ahn, Taeho; Back, Kyoungwhan

    2014-10-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 μM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

  13. Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.

    PubMed

    Neuwald, Andrew F; Altschul, Stephen F

    2016-12-01

    Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).

  14. Effect of undernutrition on the regional development of transmitter enzymes: glutamate decarboxylase and choline acetyltransferase.

    PubMed

    Patel, A J; del Vecchio, M; Atkinson, D J

    1978-01-01

    The effect of undernutrition on the activity of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAc) (markers for the GABA-ergic and the cholinergic transmitter system, respectively) was studied in various parts of the rat brain at the age of 10, 15 and 21 days, and at day 54 following 33 days of rehabilitation. The brain regions investigated were the olfactory bulbs, cerebellum, pons-medulla, hypothalamus, colliculi, cerebral cortex hippocampus and the residual brain. Undernutrition resulted in a marked retardation of the developmental rise of the activities of both enzymes, expressed in terms of either total brain part or unit weight or protein. The effect diminished with age even during the period of nutritional deprivation. In most brain regions the enzyme activities were restored to normal after rehabilitation. In the cerebral cortex the total activity of both enzymes was persistently reduced, although the concentration of GAD exceeded the control levels. A negative correlation was manifested between the activities of GAD and ChAc in the different brain parts (except the cerebellum) during development. The correlation became significant by day 21 in the controls, but only after postweaning rehabilitation of the undernourished rats. The results showed therefore that undernutrition caused a reversible retardation in the development of these two transmitter enzymes, and they suggested that even the balance of the GABA-ergic and cholinergic systems throughout the brain can be restored to normal by rehabilitation.

  15. Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300

    PubMed Central

    2015-01-01

    C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC–MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks. PMID:26985290

  16. Schizosaccharomyces pombe mst2+ Encodes a MYST Family Histone Acetyltransferase That Negatively Regulates Telomere Silencing†

    PubMed Central

    Gómez, Eliana B.; Espinosa, Joaquín M.; Forsburg, Susan L.

    2005-01-01

    Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1+ and mst2+. Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Δmst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Δmst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Δmst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression. PMID:16199868

  17. Environmental History Modulates Arabidopsis Pattern-Triggered Immunity in a HISTONE ACETYLTRANSFERASE1-Dependent Manner.

    PubMed

    Singh, Prashant; Yekondi, Shweta; Chen, Po-Wen; Tsai, Chia-Hong; Yu, Chun-Wei; Wu, Keqiang; Zimmerli, Laurent

    2014-06-01

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics.

  18. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

    PubMed

    Knowles, Joshua W; Xie, Weijia; Zhang, Zhongyang; Chennamsetty, Indumathi; Chennemsetty, Indumathi; Assimes, Themistocles L; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O; Carcamo-Orive, Ivan; Morris, Andrew P; Chen, Yii-Der I; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J; Tsao, Philip S; Schadt, Eric E; Rotter, Jerome I; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A; Groop, Leif; Cordell, Heather J; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M; Weedon, Michael N; Walker, Mark; Quertermous, Thomas

    2015-04-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

  19. Lysine acetyltransferases CBP and p300 as therapeutic targets in cognitive and neurodegenerative disorders.

    PubMed

    Valor, Luis M; Viosca, Jose; Lopez-Atalaya, Jose P; Barco, Angel

    2013-01-01

    Neuropsychiatric pathologies, including neurodegenerative diseases and neurodevelopmental syndromes, are frequently associated with dysregulation of various essential cellular mechanisms, such as transcription, mitochondrial respiration and protein degradation. In these complex scenarios, it is difficult to pinpoint the specific molecular dysfunction that initiated the pathology or that led to the fatal cascade of events that ends with the death of the neuron. Among the possible original factors, epigenetic dysregulation has attracted special attention. This review focuses on two highly related epigenetic factors that are directly involved in a number of neurological disorders, the lysine acetyltransferases CREB-binding protein (CBP) and E1A-associated protein p300 (p300). We first comment on the role of chromatin acetylation and the enzymes that control it, particularly CBP and p300, in neuronal plasticity and cognition. Next, we describe the involvement of these proteins in intellectual disability and in different neurodegenerative diseases. Finally, we discuss the potential of ameliorative strategies targeting CBP/p300 for the treatment of these disorders.

  20. Variation of the N-acetyltransferase 2 gene in a Romanian and a Kyrgyz population.

    PubMed

    Rabstein, Sylvia; Unfried, Klaus; Ranft, Ulrich; Illig, Thomas; Kolz, Melanie; Rihs, Hans-Peter; Mambetova, Chinara; Vlad, Mariana; Brüning, Thomas; Pesch, Beate

    2006-01-01

    As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3' flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomorphic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations.

  1. The Histone Acetyltransferase MOF Promotes Induces Generation of Pluripotent Stem Cells.

    PubMed

    Mu, Xupeng; Yan, Shaohua; Fu, Changhao; Wei, Anhui

    2015-08-01

    Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

  2. Muscle-specific Deletion of Carnitine Acetyltransferase Compromises Glucose Tolerance and Metabolic Flexibility

    PubMed Central

    Muoio, Deborah M.; Noland, Robert C.; Kovalik, Jean-Paul; Seiler, Sarah E.; Davies, Michael N.; DeBalsi, Karen L.; Ilkayeva, Olga R.; Stevens, Robert D.; Kheterpal, Indu; Zhang, Jingying; Covington, Jeffrey D.; Bajpeyi, Sudip; Ravussin, Eric; Kraus, William; Koves, Timothy R.; Mynatt, Randall L.

    2012-01-01

    Summary The concept of “metabolic inflexibility” was first introduced to describe the failure of insulin resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility. PMID:22560225

  3. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    SciTech Connect

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K.

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  4. Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

    PubMed

    Suganuma, Tamaki; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Workman, Jerry L

    2016-02-01

    Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation.

  5. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

    PubMed Central

    Howes, Stuart C.; Alushin, Gregory M.; Shida, Toshinobu; Nachury, Maxence V.; Nogales, Eva

    2014-01-01

    Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  6. Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse.

    PubMed

    Sadananda, Aparna; Hamid, Runa; Doodhi, Harinath; Ghosal, Debnath; Girotra, Mukul; Jana, Swadhin Chandra; Ray, Krishanu

    2012-07-01

    Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.

  7. Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars.

    PubMed

    Westwood, Isaac M; Bhakta, Sanjib; Russell, Angela J; Fullam, Elizabeth; Anderton, Matthew C; Kawamura, Akane; Mulvaney, Andrew W; Vickers, Richard J; Bhowruth, Veemal; Besra, Gurdyal S; Lalvani, Ajit; Davies, Stephen G; Sim, Edith

    2010-01-01

    New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.

  8. Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis.

    PubMed

    Takagi, Hiroshi; Yoshioka, Kenji; Awano, Naoki; Nakamori, Shigeru; Ono, Bun ichiro

    2003-01-28

    Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine.

  9. Radiosensitizing effect of the histone acetyltransferase inhibitor anacardic acid on various mammalian cell lines

    PubMed Central

    CATE, ROSEMARIE TEN; KRAWCZYK, PRZEMEK; STAP, JAN; ATEN, JACOB A.; FRANKEN, NICOLAAS A.P.

    2010-01-01

    Agents that enhance the effectiveness of ionizing radiation have been investigated over many decades. A relatively new group of potential radiosensitizers consists of agents that inhibit histone acetyltransferases (HATs). This study evaluated the radiosensitizing properties of the HAT inhibitor anacardic acid (AA), used at a low-toxic concentration of 100 μM in V79, SW1573 and U2OS cells. Radiation survival curves were analyzed according to the linear quadratic model. Significant radiosensitization by AA was only obtained in U2OS cells. AA significantly increased the value of the linear parameter α, but not of the quadratic parameter β, indicating fixation of potentially lethal damage and an intact repair function of sublethal damage. The increase of the α value was also observed in SW1573 cells, but was not accompanied by a significant radiosensitization. A likely explanation for the enhancement of the α value may be an increase in the amount of lethal lesions due to the compacted chromatin structure. Despite the conflicting results of the radiosensitizing effect of AA in the three cell lines tested, the ability of AA to increase the α value suggests potential advantages for clinical application. PMID:22966377

  10. Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

    PubMed

    Gao, Yuan; Koppen, Arjen; Rakhshandehroo, Maryam; Tasdelen, Ismayil; van de Graaf, Stan F; van Loosdregt, Jorg; van Beekum, Olivier; Hamers, Nicole; van Leenen, Dik; Berkers, Celia R; Berger, Ruud; Holstege, Frank C P; Coffer, Paul J; Brenkman, Arjan B; Ovaa, Huib; Kalkhoven, Eric

    2013-01-01

    Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

  11. Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations

    PubMed Central

    Neuwald, Andrew F.

    2016-01-01

    Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes’ theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu). PMID:28002465

  12. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey.

    PubMed

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L; Shi, Qiong

    2015-12-31

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.

  13. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  14. Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

    PubMed Central

    Huang, Fu; Paulson, Ariel; Dutta, Arnob; Venkatesh, Swaminathan; Smolle, Michaela

    2014-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. PMID:25512562

  15. Histone Acetyltransferase Complexes Can Mediate Transcriptional Activation by the Major Glucocorticoid Receptor Activation Domain

    PubMed Central

    Wallberg, Annika E.; Neely, Kristen E.; Gustafsson, Jan-Åke; Workman, Jerry L.; Wright, Anthony P. H.; Grant, Patrick A.

    1999-01-01

    Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, τ1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-τ1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in τ1 that reduce τ1 transactivation activity in vivo lead to a reduced binding of τ1 to the SAGA complex and conversely, mutations increasing the transactivation activity of τ1 lead to an increased binding of τ1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with τ1. GAL4-τ1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity τ1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity τ1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-τ1 activation domain mediates transcriptional stimulation from chromatin templates. PMID:10454542

  16. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  17. Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

    SciTech Connect

    Dairou, Julien; Petit, Emile; Ragunathan, Nilusha; Baeza-Squiban, Armelle; Marano, Francelyne; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2009-05-01

    Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and {beta}-naphthylamine ({beta}-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H{sub 2}O{sub 2} or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

  18. Crystallization and preliminary X-ray analysis of maltose O-acetyltransferase.

    PubMed

    Lo Leggio, L; Dal Degan, F; Poulsen, P; Sørensen, S O; Harlow, K; Harris, P; Larsen, S

    2001-12-01

    Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.

  19. The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network.

    PubMed

    Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

    2012-08-03

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that the histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining, and differentiation potential. They also have aberrant expression of the core transcription factors Nanog, Oct4, and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting the idea that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP-sequencing and transcriptome analyses further demonstrate that Mof is an integral component of the ESC core transcriptional network and that Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3K4 methylation at key regulatory loci, highlighting the complexity and interconnectivity of various chromatin regulators in ESCs.

  20. In vitro inhibition of choline acetyltransferase by a series of 2-benzylidene-3-quinuclidinones

    SciTech Connect

    Capacio, B.R.

    1988-01-01

    Ten substituted 2-benzylidene-3-quinuclidinones were synthesized and evaluated for their relative potency as in vitro inhibitors of choline acetyltransferase (ChAT). Acetylcholine (ACh) synthesis was followed radiometrically by the incorporation of labeled acetate originating from {sup 14}C-acetyl-CoA. Woolf-Augustinsson-Hofstee data analysis was used to calculate Vmax, Km, and Ki values. The inhibition was found to be noncompetitive or uncompetitive with respect to choline. Quantitative structure activity relationship correlations demonstrated a primary dependence on {kappa}-{sigma}, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3{prime}-methyl)-benzylidene-3-quinuclidinone, one of the more potent analogs, to further elucidate the inhibitory mechanism. ChAT-mediated cleavage of ACh was measured spectrophotometrically by following the appearance of NADH at 340 nanometers in an enzyme coupled assay. Lineweaver-Burk analysis indicated mixed or uncompetitive inhibition with respect to both substrates of the forward reaction, suggesting interference with a rate limiting step.

  1. Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.

    PubMed Central

    Morillas, Montserrat; López-VViñas, Eduardo; Valencia, Alfonso; Serra, Dolors; Gómez-Puertas, Paulino; Hegardt, Fausto G; Asins, Guillermina

    2004-01-01

    CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies. PMID:14711372

  2. Undrained shear strength of partially saturated combined coal refuse. First annual report: Strength and consolidation characteristics of coal refuse for design and construction of disposal facilities

    SciTech Connect

    Huang, Y.H.; Li, J.

    1986-09-01

    This report summarizes the results of a study on the undrained shear strength of partially saturated combined refuse. The study is part of a research project entitled 'Strength and Consolidation Characteristics of Coal Refuse for Design and Construction of Disposal Facilities supported by the Office of Surface Mining, Department of the Interior. Information presented in the report will be used for the design and construction of disposal facilities. Coal refuse, the waste product from coal washing, is separated in the coal preparation plant into two fractions (coarse and fine). The fine refuse, in the form of either a slurry or a filter cake, is unstable and difficult to handle.

  3. Review of Programs: Architecture; Architectural Technology; Landscape Architecture; Interior Design; Construction and Construction Technology; Building Construction; Urban and Regional Planning. Report to the Board of Regents, State University System of Florida.

    ERIC Educational Resources Information Center

    McMinn, William G.

    This report is an update of a report on the development and status of various programs in architecture and related fields in the State University System of Florida, a report that was submitted to the Board of Regents in May 1983. The objectives of this updated report, like those of the earlier one, are to review the anticipated needs of the…

  4. State of the practice: Design and construction of asphalt paving materials with crumb-rubber modifier. Final report

    SciTech Connect

    Heitzman, M.A.

    1992-05-01

    The document is a comprehensive overview of the terminology, processes, products, and applications of crumb rubber modifier (CRM) technology. The technology includes any use of scrap tire rubber in asphalt paving materials. In general, CRM technology can be divided into two categories--the wet process and the dry process. When CRM is incorporated into an asphalt paving material, it will modify the properties of the binder (asphalt rubber) and/or act as a rubber aggregate (rubber modified hot mix asphalt). The five concepts for using CRM discussed in the report are McDonald, PlusRide, generic dry, chunk rubber asphalt concrete, and continuous blending asphalt rubber. There are two principal unresolved engineering issues related to the use of CRM in asphalt paving materials. On the national level, the ability to recycle asphalt paving mixes containing CRM has not been demonstrated. At the State and local levels, these modified asphalt mixes must be field evaluated to establish expected levels of performance. The appendices provide guidelines for material specifications, mix design, and construction specifications. An experimental work plan for monitoring performance and a stack emission testing program are also included.

  5. Construction of an implant-retained auricular prosthesis with the aid of contemporary digital technologies: a clinical report.

    PubMed

    Hatamleh, Muhanad M; Watson, Jason

    2013-02-01

    Implant-retained auricular prostheses are a successful treatment modality for children with microtia. They involve only minor surgical intervention of implant placement and result in an esthetically pleasing outcome. Integration of digital technologies (DT) in the prosthetic reconstruction process is a new approach toward enhancing outcomes. In this report we present a case of auricular prosthetic reconstruction following two implant placements in the right mastoid region. The ear prosthesis was constructed with the aid of various DTs. A structured light laser scanner was used to digitize the nondefect patient ear. The digitized 3D ear was then manipulated in specialist software, mirrored to reflect the opposing side, and a Rapid Prototyping (RP) machine (Z-Corp) was used to manufacture the soft tissue required. This RP-mirrored ear model allows very accurate reproduction to replicate missing soft tissue. A color Spectrometer was used to accurately reproduce skin tones. The use of these technologies is now routine practice at our unit. They enhance prosthetic outcomes and esthetics, save the prosthetist's time, and are digitally stored and subsequently readily available and reproducible.

  6. Evaluation and utilization of Illinois FBC residues for construction materials. Technical progress report, March 1, 1992--May 31, 1992

    SciTech Connect

    Ghafoori, N.; Sami, S.

    1992-10-01

    The overall objective of this program is to investigate the extent to which fluidized bed combustion (FBC) by-products can be properly utilized as viable construction materials. This investigation focuses primarily on the properties of residues derived from fluidized combustion burning of Illinois high-sulfur coal. The research plan calls for evaluation of physics-chemical and engineering characteristics of the FBC-based cement and non-cement mixes. The results of this study will be used to compare the physical and mechanical properties of the FBC-based mixtures with those of conventional mixes. The suitability of using FBC residues as a filler or binder aggregate for Portland cement-based mixtures and Non-Portland cement mixes in the form of conventional and roller compacted materials will be evaluated. During this reporting period, physics-chemical properties of class F fly ash, supplied by the American Fly Ash Company of Naperville, Illinois, were completed. Work on all major tasks, sulfate durability of FBC cement-based mixtures and Engineering characteristics of roller compacted non-cement FBC mixes (task II), are on-going.

  7. Evaluation and utilization of Illinois FBC residues for construction materials. Final technical report, September 1, 1991--August 31, 1992

    SciTech Connect

    Ghafoori, N.; Sami, S.

    1992-12-31

    The overall objective of this program is to investigate the extent to which fluidized bed combustion (FBC) by-products can be properly utilized as viable construction materials. This investigation focuses primarily on the properties of residues derived from fluidized combustion burning of Illinois high-sulfur coal. The research plan calls for evaluation of physics-chemical and engineering characteristics of the FBC-based cement and non-cement mixes. The results of this study will be used to compare the physical and mechanical properties of the FBC-based mixtures with those of conventional mixes. The suitability of using FBC residues as filler or binder aggregates for Portland cement-based mixtures and non-Portland cement mixes in the form of conventional and roller compacted materials will be evaluated. During this reporting period, fabrication of the laboratory specimens used for evaluation of the sulfate durability of FBC cement-based mixtures (task I) were completed. Work on engineering characteristics of roller compacted non-cement FBC mixes (task II), is on-going.

  8. Conceptual Design Report: Fermilab Main Injector - Technical Components and Civil Construction, April 1992 (Rev. 3.1)

    SciTech Connect

    1992-04-01

    This report contains a description of the design and cost estimate of a new 150 GeV accelerator, designated the Fermilab Main Injector (FMI). The construction of this accelerator will simultaneously result in significant enhancements to both the Fermilab collider and fixed target programs. The FMI is to be located south of the Antiproton Source and tangent to the Tevatron ring at the FO straight section as shown in Figure 1-1. The FMI will perform all duties currently required of the existing Main Ring. Thus, operation of the Main Ring will cease following commissioning of the FMI, with a concurrent reduction in background rates as seen in the colliding beam detectors. The performance of the FMI, as measured in terms of protons per second delivered to the antiproton production target or total protons delivered to the Tevatron, is expected to exceed that of the Main Ring by a factor of two-tothree. In addition the FMI will provide high duty factor 120 GeV beam to the experimental areas during collider operation, a capability which does not presently exist in the Main Ring.

  9. Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli.

    PubMed

    Kataoka, Naoya; Vangnai, Alisa S; Pongtharangkul, Thunyarat; Tajima, Takahisa; Yakushi, Toshiharu; Matsushita, Kazunobu; Kato, Junichi

    2015-06-20

    1-Butanol is an important industrial platform chemical and an advanced biofuel. While various groups have attempted to construct synthetic pathways for 1-butanol production, efforts to construct a pathway that functions under aerobic conditions have met with limited success. Here, we constructed a CoA-dependent 1-butanol synthetic pathway that functions under aerobic conditions in Escherichia coli, by expanding the previously reported (R)-1,3-butanediol synthetic pathway. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, bld (butylraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and inherent alcohol dehydrogenase(s) from E. coli. To evaluate the potential of this pathway for 1-butanol production, culture conditions, including volumetric oxygen transfer coefficient (kLa) and pH were optimized in a mini-jar fermenter. Under optimal conditions, 1-butanol was produced at a concentration of up to 8.60gL(-1) after 46h of fed-batch cultivation.

  10. Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.

    PubMed

    Ainsworth, W B; Rome, C M; Hjortsø, M A; Benton, M G

    2012-12-01

    Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.

  11. Radon reduction and radon-resistant construction demonstrations in New York. Volume 1. Technical report. Final report, March 1987-February 1991

    SciTech Connect

    Nitschke, I.; Clarkin, M.; Clark, W.; Hough, R.E.

    1993-03-01

    The report gives results of radon reduction and radon-resistant construction demonstrations in New York. The existing house evaluation demonstrated radon mitigation techniques where indoor radon concentrations exceeded 4 pCi/L. Results demonstrated that sealing all accessible foundation penetrations in the basement effectively reduced the radon concentration, although not below 4 pCi/L, and that sealing aids the effectiveness of an active depressurization system. Active depressurization systems were usually successful in achieving 4 pCi/L. The footing drain, sub-slab, and basement walls were all successfully depressurized using a standard technique after grab samples or radon 'sniffing' techniques were used to identify the radon entry sources. Basement pressurization also effectively reduced the radon level below the EPA guideline at one site. Water aeration systems effectively mitigated radon from residential water supplies, although the system tested was large and noisy. Activated charcoal filters adsorbed the radon in water, but eventually became an unacceptable source of gamma radiation. The inspection of houses where radon mitigation systems were installed in 1984 revealed that new systems and techniques, such as in-line centrifugal fans, were generally superior to the earlier methods using axial computer-type fans.

  12. Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

    PubMed Central

    Selvi, Ruthrotha B.; Swaminathan, Amrutha; Chatterjee, Snehajyoti; Shanmugam, Muthu K.; Li, Feng; Ramakrishnan, Gowsica B.; Siveen, Kodappully Sivaraman; Chinnathambi, Arunachalam; Zayed, M. Emam; Alharbi, Sulaiman Ali; Basha, Jeelan; Bhat, Akshay; Vasudevan, Madavan; Dharmarajan, Arunasalam; Sethi, Gautam; Kundu, Tapas K.

    2015-01-01

    Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing. PMID:26517526

  13. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

    PubMed

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

  14. Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT) gene.

    PubMed

    Feldhammer, Matthew; Durand, Stéphanie; Mrázová, Lenka; Boucher, Renée-Myriam; Laframboise, Rachel; Steinfeld, Robert; Wraith, James E; Michelakakis, Helen; van Diggelen, Otto P; Hrebícek, Martin; Kmoch, Stanislav; Pshezhetsky, Alexey V

    2009-06-01

    Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.

  15. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1)

    PubMed Central

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes. PMID:28280335

  16. Yng1p Modulates the Activity of Sas3p as a Component of the Yeast NuA3 Histone Acetyltransferase Complex

    PubMed Central

    Howe, LeAnn; Kusch, Thomas; Muster, Nemone; Chaterji, Ranjana; Yates III, John R.; Workman, Jerry L.

    2002-01-01

    The mammalian ING1 gene encodes a tumor suppressor required for the function of p53. In this study we report a novel function for YNG1, a yeast homolog of ING1. Yng1p is a stable component of the NuA3 histone acetyltransferase complex, which contains Sas3p, the yeast homolog of the mammalian MOZ proto-oncogene product, as its catalytic subunit. Yng1p is required for NuA3 function in vivo but surprisingly is not required for the integrity of the complex. Instead, we find that Yng1p mediates the interaction of Sas3p with nucleosomes and is thus required for the ability of NuA3 to modify histone tails. These data, and the observations that other ING1 homologs are found in additional yeast complexes that posttranslationally modify histones, suggest that members of the ING1 class of proteins may have broad roles in enhancing or modifying the activities of chromatin-modifying complexes, thereby regulating their activities in transcription control. PMID:12077334

  17. Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

    PubMed Central

    Barlev, Nickolai A.; Poltoratsky, Vladimir; Owen-Hughes, Tom; Ying, Carol; Liu, Lin; Workman, Jerry L.; Berger, Shelley L.

    1998-01-01

    GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity. PMID:9488450

  18. Generation IV Nuclear Energy Systems Construction Cost Reductions through the Use of Virtual Environments - Task 4 Report: Virtual Mockup Maintenance Task Evaluation

    SciTech Connect

    Timothy Shaw; Anthony Baratta; Vaughn Whisker

    2005-02-28

    Task 4 report of 3 year DOE NERI-sponsored effort evaluating immersive virtual reality (CAVE) technology for design review, construction planning, and maintenance planning and training for next generation nuclear power plants. Program covers development of full-scale virtual mockups generated from 3D CAD data presented in a CAVE visualization facility. This report focuses on using Full-scale virtual mockups for nuclear power plant training applications.

  19. School Facilities Construction Expenditures Have Grown Significantly in Recent Years. Report to the Chairman, Committee on Education and the Workforce, House of Representatives.

    ERIC Educational Resources Information Center

    Bovbjerg, Barbara D.

    This report examines how states and local school districts have been dealing with the issues facing their public school facilities, including: (1) the trends since 1990 in elementary and secondary school construction expenditures and how these expenditures were divided between land, buildings, and equipment; (2) trends since 1990 in the amount of…

  20. Parent- and Self-Reported Dimensions of Oppositionality in Youth: Construct Validity, Concurrent Validity, and the Prediction of Criminal Outcomes in Adulthood

    ERIC Educational Resources Information Center

    Aebi, Marcel; Plattner, Belinda; Metzke, Christa Winkler; Bessler, Cornelia; Steinhausen, Hans-Christoph

    2013-01-01

    Background: Different dimensions of oppositional defiant disorder (ODD) have been found as valid predictors of further mental health problems and antisocial behaviors in youth. The present study aimed at testing the construct, concurrent, and predictive validity of ODD dimensions derived from parent- and self-report measures. Method: Confirmatory…

  1. Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in Rhipicephalus (Boophilus) microplus cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  2. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    SciTech Connect

    Oike, Takahiro; Ogiwara, Hideaki; Torikai, Kohta; Nakano, Takashi; Yokota, Jun; Kohno, Takashi

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  3. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity.

    PubMed

    Stimson, Lindsay; Rowlands, Martin G; Newbatt, Yvette M; Smith, Nicola F; Raynaud, Florence I; Rogers, Paul; Bavetsias, Vassilios; Gorsuch, Stephen; Jarman, Michael; Bannister, Andrew; Kouzarides, Tony; McDonald, Edward; Workman, Paul; Aherne, G Wynne

    2005-10-01

    Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

  4. CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells

    PubMed Central

    Sauer, M; Schuldner, M; Hoffmann, N; Cetintas, A; Reiners, K S; Shatnyeva, O; Hallek, M; Hansen, H P; Gasser, S; von Strandmann, E P

    2017-01-01

    Tumor surveillance of natural killer (NK) cells is mediated by the cytotoxicity receptor natural-killer group 2 member D (NKG2D). Ligands for NKG2D are generally not expressed on healthy cells, but induced on the surface of malignant cells. To date, NKG2D ligand (NKG2D-L) induction was mainly described to depend on the activation of the DNA damage response, although the molecular mechanisms that regulate NKG2D-L expression remain largely unknown. Here, we show that the acetyltransferases CBP (CREB-binding protein) and p300 play a crucial role in the regulation of NKG2D-L on tumor cells. Loss of CBP/p300 decreased the basal cell surface expression of human ligands and reduced the upregulation of MICA/B and ULBP2 in response to histone deacetylase inhibitors or DNA damage. Furthermore, CBP/P300 deficiency abrogated the sensitivity of stressed cells to NK cell-mediated killing. CBP/p300 were also identified as major regulators of mouse NKG2D ligand RAE-1 in vitro and in vivo using the Eμ-Myc lymphoma model. Mechanistically, we observed an enhanced activation of the CBP/p300 binding transcription factor CREB (cAMP response element-binding protein) correlating to the NKG2D-L upregulation. Moreover, increased binding of CREB and CBP/p300 to NKG2D-L promoters and elevated histone acetylation were detectable. This study provides strong evidence for a major role of CBP and p300 in orchestrating NKG2D-L induction and consequently immunosurveillance of tumors in mice and humans. These findings might help to develop novel immunotherapeutic approaches against cancer. PMID:27477692

  5. Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase

    PubMed Central

    Nishino, Taito; Wang, Changshan; Mochizuki-Kashio, Makiko; Osawa, Mitsujiro; Nakauchi, Hiromitsu; Iwama, Atsushi

    2011-01-01

    Background Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. Methodology/Principal Findings In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34+CD38– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34+CD38– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34+ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. Conclusions/Significance Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. PMID:21931675

  6. Melatonin and serotonin N-acetyltransferase activity in developing eggs of the cricket Gryllus bimaculatus.

    PubMed

    Itoh, M T; Sumi, Y

    1998-01-19

    Melatonin (N-acetyl-5-methoxytryptamine) and serotonin N-acetyltransferase (NAT), a key regulatory enzyme in melatonin synthesis, are present in the adults and larvae of several insect species, as well as in vertebrates. To determine when melatonin and NAT first appear in insects ontogenetically, melatonin levels and NAT-like activity were measured in developing eggs of the cricket Gryllus bimaculatus. When the eggs were incubated under a 12-h light/12-h dark (LD) cycle at 24-26 degrees C, melatonin was detected in the egg extracts at all of the developmental stages examined. NAT-like activity was first found in the eggs 3 days after oviposition. From 5 to 11 days after oviposition, both NAT-like activity and melatonin levels showed significant day/night changes with the high levels occurring during the dark period of the LD cycle. By contrast, significant day/night changes were not detected in eggs just before hatching. To determine more detailed temporal changes, NAT-like activity was assayed in eggs 6 to 7 days after oviposition at 2- or 4-h intervals over a 48-h period. The activity in the eggs clearly exhibited a diurnal rhythm, peaking in the dark period of the LD cycle, and the rhythm persisted in constant darkness. These results suggest that the cricket egg (probably the embryo) synthesizes melatonin, and that its melatonin synthesis may fluctuate with a circadian rhythm. In addition, the results of the present study strongly suggest that a circadian clock controlling NAT activity functions in the cricket at the embryonic stage.

  7. Ocimum sanctum Linn. stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells

    PubMed Central

    Kusindarta, Dwi Liliek; Wihadmadyatami, Hevi; Haryanto, Aris

    2016-01-01

    Aim: This research was conducted to identify the expression of choline acetyltransferase (ChAT) in human cerebral microvascular endothelial cells (HCMECs) and to clarify the capability of Ocimum sanctum Linn. ethanolic extract to stimulate the presence of ChAT in the aging HCMECs. Materials and Methods: In this study, we perform an in vitro analysis some in the presence of an ethanolic extract of O. sanctum Linn. as a stimulator for the ChAT expression. HCMECs are divided become two groups, the first is in low passage cells as a model of young aged and the second is in a high passage as a model of aging. Furthermore to analysis the expression of ChAT without and with extract treatments, immunocytochemistry and flow cytometry analysis were performed. In addition, ChAT sandwich enzyme-linked immunosorbent assay is developed to detect the increasing activity of the ChAT under normal, and aging HCMECs on the condition treated and untreated cells. Results: In our in vitro models using HCMECs, we found that ChAT is expressed throughout intracytoplasmic areas. On the status of aging, the ethanolic extract from O. sanctum Linn. is capable to stimulate and restore the expression of ChAT. The increasing of ChAT expression is in line with the increasing activity of this enzyme on the aging treated HCMECs. Conclusions: Our observation indicates that HCMECs is one of the noncholinergic cells which is produced ChAT. The administrated of O. sanctum Linn. ethanolic extract may stimulate and restore the expression of ChAT on the deteriorating cells of HCMECs, thus its may give nerve protection and help the production of acetylcholine. PMID:28096604

  8. Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

    PubMed Central

    Sim, E; Abuhammad, A; Ryan, A

    2014-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic drug-metabolizing enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. The slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The two polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite para-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in oestrogen receptor-positive breast cancer and may contribute to folate and acetyl CoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site-modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetyl CoA and differs among NATs including prokaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolizes isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly, nat is in a gene cluster essential for Mycobacterium tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculosis drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5-aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide. PMID:24467436

  9. Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

    PubMed

    Kobayashi, Jyumpei; Furukawa, Megumi; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-07-01

    Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

  10. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  11. Influence of photoperiod on N-acetyltransferase activity and melatonin in the fiddler crab Uca pugilator.

    PubMed

    Tilden, A R; Alt, J; Brummer, K; Groth, R; Herwig, K; Wilson, A; Wilson, S

    2001-06-01

    Melatonin and N-acetyltransferase (NAT) activity were measured in the eyestalks of fiddler crabs acclimated to various photoperiods: constant light, a L:D 12:12 h photoperiod, or constant dark. Following acclimation, eyestalks were collected every 3 h over a 24-h period; they were assayed for melatonin with a radioimmunoassay and for NAT activity with a radioenzymatic assay. In constant light, melatonin levels increased at 1300 h, from 142 to 431 pg x mg(-1) eyestalk; NAT activity increased concurrently, from 97 to 203 pmol x h(-1) x mg(-1) eyestalk, and both remained elevated until 0400 h. In the L:D 12:12 h photoperiod, melatonin levels increased at 1300 h from 28 to 230 pg x mg(-1) eyestalk, and though NAT activity increased significantly, from 80 to 122 pmol x h(-1) x mg(-1) eyestalk, an even greater increase occurred at 0400 h, when melatonin levels were low. In constant dark, melatonin levels increased at 1600 h, from 22 to 196 pg x mg(-1) eyestalk, with a concurrent increase in NAT activity from 93 to 140 pmol x mg(-1) x h(-1) eyestalk. However, the second peak in melatonin (111 pg x mg(-1)), occurring at 0400 h, was out of phase with the second peak of NAT activity (113 pmol x mg(-1) x h(-1) eyestalk) which occurred at 0700 h. NAT may be a rate-limiting step in melatonin synthesis in fiddler crabs under some conditions (constant light and the 1300 h peak in constant dark); however, NAT activity correlates poorly with melatonin levels in a L:D 12:12 h photoperiod and in constant dark relative to the 0400 h melatonin peak.

  12. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer.

    PubMed

    Mahasneh, Amjad; Jubaili, Amal; El Bateiha, Ahmed; Al-Ghazo, Mohammad; Matalka, Ismail; Malkawi, Mousa

    2012-12-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.

  13. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

    PubMed Central

    Mahasneh, Amjad; Jubaili, Amal; El Bateiha, Ahmed; Al-Ghazo, Mohammad; Matalka, Ismail; Malkawi, Mousa

    2012-01-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan. PMID:23271930

  14. N-acetyltransferase 2, exposure to aromatic and heterocyclic amines, and receptor-defined breast cancer.

    PubMed

    Rabstein, Sylvia; Brüning, Thomas; Harth, Volker; Fischer, Hans-Peter; Haas, Susanne; Weiss, Tobias; Spickenheuer, Anne; Pierl, Christiane; Justenhoven, Christina; Illig, Thomas; Vollmert, Caren; Baisch, Christian; Ko, Yon-Dschun; Hamann, Ute; Brauch, Hiltrud; Pesch, Beate

    2010-03-01

    The role of N-acetyltransferase 2 (NAT2) polymorphism in breast cancer is still unclear. We explored the associations between potential sources of exposure to aromatic and heterocyclic amines (AHA), acetylation status and receptor-defined breast cancer in 1020 incident cases and 1047 population controls of the German GENICA study. Acetylation status was assessed as slow or fast. Therefore, NAT2 haplotypes were estimated using genotype information from six NAT2 polymorphisms. Most probable haplotypes served as alleles for the deduction of NAT2 acetylation status. The risks of developing estrogen receptor alpha (ER) and progesterone receptor (PR)-positive or negative tumors were estimated for tobacco smoking, consumption of red meat, grilled food, coffee, and tea, as well as expert-rated occupational exposure to AHA with logistic regression conditional on age and adjusted for potential confounders. Joint effects of these factors and NAT2 acetylation status were investigated. Frequent consumption of grilled food and coffee showed higher risks in slow acetylators for receptor-negative tumors [grilled food: ER-: odds ratio (OR) 2.57, 95% confidence interval (CI) 1.07-6.14 for regular vs. rare; coffee: ER-: OR 2.55, 95% CI 1.22-5.33 for >or=4 vs. 0 cups/day]. We observed slightly higher risks for never smokers that are fast acetylators for receptor-positive tumors compared with slow acetylators (ER-: OR 1.32, 95% CI 1.00-1.73). Our results support differing risk patterns for receptor-defined breast cancer. However, the modifying role of NAT2 for receptor-defined breast cancer is difficult to interpret in the light of complex mixtures of exposure to AHA.

  15. Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

    PubMed

    Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

    2017-02-15

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1-10. ©2017 AACR.

  16. Spatiotemporal expression of histone acetyltransferases, p300 and CBP, in developing embryonic hearts

    PubMed Central

    Chen, Guozhen; Zhu, Jing; Lv, Tiewei; Wu, Gang; Sun, Huichao; Huang, Xupei; Tian, Jie

    2009-01-01

    Histone acetyltransferases (HATs), p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes involved in cellular growth and signaling, muscle differentiation and embryogenesis. However, whether these proteins play important and different roles in mouse cardiogenesis is not clear. Here, we investigate the protein distributions and mRNA expression of the two HATs in embryonic and adult mouse heart during normal heart development by using immunohistochemical and RT-PCR techniques. The data from immunohistochemical experiments revealed that p300 was extensively present in nearly every region of the hearts from embryonic stages to the adulthood. However, no CBP expression was detected in embryonic hearts at day E7.5. CBP expression appeared at the later stages, and the distribution of CBP was less than that of p300. In the developmental hearts after E10.5, both for p300 and CBP, the mRNA expression levels reached a peak on day E10.5, and then were gradually decreased afterwards. These results reveal that both p300 and CBP are related to embryonic heart development. The dynamic expression patterns of these two enzymes during mouse heart development indicate that they may play an important role on heart development. However, there is a difference in spatiotemporal expression patterns between these two enzymes during heart development. The expression of p300 is earlier and more predominate, suggesting that p300 may play a more important role in embryonic heart development especially during cardiac precursor cell induction and interventricular septum formation. PMID:19272189

  17. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    SciTech Connect

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  18. Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype-Phenotype Correlations.

    PubMed

    Arredondo, Juan; Lara, Marian; Gospe, Sídney M; Mazia, Claudio G; Vaccarezza, Maria; Garcia-Erro, Marcela; Bowe, Constance M; Chang, Celia H; Mezei, Michelle M; Maselli, Ricardo A

    2015-09-01

    Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype-phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate-binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.

  19. Choline acetyltransferase mutations causing congenital myasthenic syndrome: molecular findings and genotype-phenotype correlations

    PubMed Central

    Arredondo, Juan; Lara, Marian; Gospe, Sídney M.; Mazia, Claudio G.; Vaccarezza, Maria; Garcia-Erro, Marcela; Bowe, Constance; Chang, Celia; Mezei, Michelle; Maselli, Ricardo A.

    2015-01-01

    Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome (CMS) due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype–phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of seven ChAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal instability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met is located far from both active and substrate-binding sites produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes. PMID:26080897

  20. Epigenetic regulation of spermidine/spermine N1-acetyltransferase (SAT1) in suicide.

    PubMed

    Fiori, Laura M; Turecki, Gustavo

    2011-09-01

    We have recently shown that the expression of spermidine/spermine N1-acetyltransferase (SAT1) is downregulated across the brains of suicide completers, and that its expression is influenced by genetic variations in the promoter. Several promoter polymorphisms in SAT1, including rs6526342, have been associated with suicide and other psychiatric disorders, and display haplotype-specific effects on expression. However, these effects cannot explain total variability in SAT1 expression, and other regulatory mechanisms, such as epigenetic factors, may also be at play. In this study, we assessed the involvement of epigenetic factors in controlling SAT1 expression in the prefrontal cortex of suicide completers by mapping CpG methylation across a 1880-bp region of the SAT1 promoter, and measuring levels of tri-methylated histone-3-lysine 27 (H3K27me3) at the promoter in suicide completers and controls. Our results demonstrated that CpG methylation was significantly negatively correlated with SAT1 expression. Although overall or site-specific CpG methylation was not associated with suicide or SAT1 expression, we observed high levels of methylation at the polymorphic CpG site created by rs6526342, indicating a relationship between promoter haplotypes and methylation. There was no association between H3K27me3 and suicide, nor was this modification associated with SAT1 expression. Overall, our results indicate that epigenetic factors in the promoter region of SAT1 influence gene expression levels, and may provide a mechanism for both our previous findings of haplotype-specific effects of promoter variations on SAT1 expression, as well as the widespread downregulation of SAT1 expression observed in the brains of suicide completers.

  1. Molecular and Biochemical Analysis of a Madagascar Periwinkle Root-Specific Minovincinine-19-Hydroxy-O-Acetyltransferase1

    PubMed Central

    Laflamme, Pierre; St-Pierre, Benoit; De Luca, Vincenzo

    2001-01-01

    The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances. PMID:11154328

  2. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

    PubMed Central

    Durrett, Timothy P.; McClosky, Daniel D.; Tumaney, Ajay W.; Elzinga, Dezi A.; Ohlrogge, John; Pollard, Mike

    2010-01-01

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications. PMID:20439724

  3. Workshops and incentive loans program for construction of solar greenhouses, grain dryers, window boxes and food dryers. Final performance report

    SciTech Connect

    Walker, D.H.

    1982-02-01

    A Solar Loan Fund was established to provide low-interest incentive loans to families desiring to construct their own solar devices, the only stipulations being allowance of reasonable public visitation and monitoring and employment of parish-trained construction teams if labor is to be hired. About 30 vocational agriculture students built window box collector units as part of their classroom experience, trying several designs. Later, 12 to 15 families built window box collectors for their use. A week long Energy Responsibility: Today and Tomorrow workshop was held involving construction projects and mini-seminars. Another workshop was held to construct bread box type solar hot water heaters, and yet another to build solar food dryers. (LEW)

  4. New developments at Hunveyor and Husar space probe model constructions in Hungarian Universities and Colleges: status report of 2008

    NASA Astrophysics Data System (ADS)

    Hegzi, S.; Bérczi, Sz.; Hudoba, Gy.; Magyar, I.; Lang, A.; Istenes, Z.; Weidinger, T.; Tepliczky, I.; Varga, T.; Hargitai, H.

    2008-09-01

    Introduction Hunveyor and Husar space probe models are the main school robotics program in Hungary in the last decade initiated by our Cosmic Materials Space research Group (CMSRG). As a new form of planetary science education in Hungary students build their lander and rover robots and test them on test tables, carry out simulations, and go with their instruments to field works of planetary geology analog sites. Recently 10 groups work in this program and here is a status report about the new results. Planetary robot construction and simulations steps We summarized in 10 steps the main "constructional and industrial research and technology" description of planetary material studying and collecting by space probes (landers, rovers). We focused on the activity we began and teach to carry out at those steps. (Main planets considered were the Moon and Mars): 1. Reconnaissance and survey of the surface of a planet by orbital space probes (i.e. Lunar Orbiter, MGS, MRO etc.) Our studies: photogeology, geomorphology, preparations to cartography. 2. Mapping of the surface of the selected planet with geographical and stratigraphical methods. We (CMSRG) prepared thematic maps on Moon, Mercury, Mars, Venus [1] and Atlas (3) in the series [2,3]. 3. Identification of various surface materials by albedo, spectroscopic [4], thermal IR, identification and selection of the target sites. (in terrestrial analog sites during field works) 4. Planning the space probe system lander and rover working together (MPF-Sojourner type assembly). Planning of the Hunveyor and Husar models. 5. Construction and manufacturing lander and rover units. All Hunveyor groups built their models [5]. 6. Launching and traveling the space probes to the planetary surface. (No rocket building, we simulate [6] some events during the voyage only). 7. Measuring the planetary surface environment on the surface of target planet [7]. (CMSRG) groups carry out test-table measurements [8] and simulations, and later they

  5. Tumor necrosis factor alpha induces spermidine/spermine N1-acetyltransferase through nuclear factor kappaB in non-small cell lung cancer cells.

    PubMed

    Babbar, Naveen; Hacker, Amy; Huang, Yi; Casero, Robert A

    2006-08-25

    Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

  6. aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed

    Rather, P N; Solinsky, K A; Paradise, M R; Parojcic, M M

    1997-04-01

    The 2'-N-acetyltransferase [AAC(2')-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2')-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2')-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2')-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein. The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

  7. Leptin-induced Growth Stimulation of Breast Cancer Cells Involves Recruitment of Histone Acetyltransferases and Mediator Complex to CYCLIN D1 Promoter via Activation of Stat3*

    PubMed Central

    Saxena, Neeraj K.; Vertino, Paula M.; Anania, Frank A.; Sharma, Dipali

    2010-01-01

    Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes. PMID:17344214

  8. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

    PubMed Central

    Evans, D J; Jones, R; Woodley, P R; Wilborn, J R; Robson, R L

    1991-01-01

    Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. PMID:1885524

  9. Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitroreductase activities

    SciTech Connect

    Oda, Yoshimitsu; Yamazaki, Hiroshi; Watanabe, Masahiko; Nohmi, Takehiko; Shimada, Tsutomu )

    1993-01-01

    A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high O-acetyltransferase (O-AT) and nitroreductase (NR) activities. The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYC184, and the resulting plasmid was introduced into the parent tester strain S. typhimurium TA1535/pSK1002 harboring an umuC[prime]-[prime]lacZ fusion gene. The induction of umuC gene expression could be monitored by measuring the cellular [beta]-galactosidase activity produced by fusion gene. The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds. The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpressing strain NM2009, and the O-AT-defective strain NM2000. The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S. typhimurium TA1535/pSK1002 strain. Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1-nitronaphthalene, 2-nitrofluorene, 3,7-dinitro-fluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4[prime]-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene. The authors have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000. 40 refs., 3 figs., 3 tabs.

  10. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    SciTech Connect

    He, Yuan; Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J.

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  11. Inhibition of cytosolic human forebrain choline acetyltransferase activity by phospho-L-serine: a phosphomonoester that accumulates during early stages of Alzheimer's disease.

    PubMed

    Andriamampandry, C; Kanfer, J N

    1993-01-01

    There is no satisfactory explanation for the cholinergic deficit characteristic of Alzheimer's disease. We have performed a series of experiments which demonstrate that (a) an inhibitor of cytosolic human brain choline acetyltransferase is present in the cytosol of Alzheimer brain tissue, (b) human brain cytosolic choline acetyltransferase activity is inhibited by phospho-L-serine in a competitive manner. Cytosol was prepared from human forebrain or amygdala and the Km for choline and acetyl CoA of the choline acetyltransferase were 750 microM and 12.5 microM, respectively. Phospho-L-serine was found to be a competitive inhibitor of this enzyme with respect to choline but not with respect to acetyl CoA with a Ki of 750 microM for the human forebrain and 3 mM for human amygdala. These concentrations of phospho-L-serine are present in brain tissue at early stages of Alzheimer's disease. Several other phosphomonoesters and phosphodiesters that are increased in Alzheimer's disease were either less inhibitory or without effect. The addition of heat denatured and non-heat denatured cytosol from Alzheimers forebrain inhibited the choline acetyltransferase activity present in control human brain cytosol. The inhibitory activity of the Alzheimers cytosol was retained in TCA deproteinized samples and removed by dialysis or by alkaline phosphatase treatment. Dialysis of the cytosol increased the choline acetyltransferase activity of 5 of 8 Alzheimer's disease samples from 21 to 118% with p values of < 0.025 or < 0.001, respectively. These observations provide evidence that an endogenous non-proteinaceous, dialyzable, phosphomonoester, present in Alzheimers brain inhibits the choline acetyltransferase of both control and Alzheimers brain.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

    PubMed

    Foy, Rebecca L; Song, Ihn Young; Chitalia, Vipul C; Cohen, Herbert T; Saksouk, Nehme; Cayrou, Christelle; Vaziri, Cyrus; Côté, Jacques; Panchenko, Maria V

    2008-10-24

    Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

  13. Association between polymorphisms at N-acetyltransferase 1 (NAT1) & risk of oral leukoplakia & cancer

    PubMed Central

    Majumder, Mousumi; Ghosh, Saurabh; Roy, Bidyut

    2012-01-01

    Background & objectives: N-acetyltransferases 1 and 2 (NAT1 and NAT2) are important enzymes for metabolism of tobacco carcinogens. Due to polymorphisms, improper activities of these enzymes might lead to the formation of DNA adducts that may modulate risk of tobacco related oral precancer and cancer. Previously, it was shown that NAT2 polymorphisms did not modulate the risk of oral precancer and cancer. We undertook this study to check whether polymorphisms at NAT1 can modulate the risk of oral leukoplakia and cancer either alone or in combination with NAT2. Methods: Genotypes at four SNPs on NAT1 were determined by TaqMan method in 389 controls, 224 leukoplakia and 310 cancer patients. Genotype data were analyzed to know haplotypes and acetylation status of individuals and, then to estimate the risk of diseases. Using our previously published NAT2 data, combination of NAT1 and NAT2 acetylation genotypes of patients and controls were also analyzed to estimate the risk of diseases. Results: Analysis of NAT1 genotype data revealed that 1088T and 1095C alleles exist in strong linkage disequilibrium (r2=0.97, P<0.0001) and SNPs are in Hardy-Weinberg Equilibrium (P=0.1). Wild type or normal acetylating and variant or rapid acetylating alleles were two major alleles (frequencies 0.62 and 0.36, respectively) present in the control population. NAT1 rapid acetylation could not modulate the risk of leukoplakia and cancer (OR=0.9, 95% CI: 0.6-1.3; OR=1.0, 95% CI: 0.7-1.4, respectively). Analysis of combined NAT1 and NAT2 acetylating data also showed no significant enhancement of the risk of diseases. Interpretation & conclusions: NAT1 rapid acetylation alone as well as combination of NAT1 rapid-NAT2 slow acetylation did not modulate the risk of oral precancer and cancer in our patient population. So, NAT1/NAT2 metabolized carcinogen products may not be involved in tobacco related oral precancer and cancer. It may be interpreted that large sample size as well as combination of

  14. DOE small-scale hydroelectric demonstration project: Riegel Textile Corporation, Fries, Virginia plant hydroproject. Final technical and construction cost report

    SciTech Connect

    Not Available

    1982-06-01

    The Riegel Textile Corporation completed a 2163 kW generator project at its plant in Fries, Virginia. A new powerhouse was constructed to enclose a used 2900 hp vertical Kaplan turbine and Westinghouse generator. Construction was accomplished without modification to or rehabilitation of an existing dam and required only minor modification to or rehabilitation of an existing dam and required only minor modification to the existing appurtenances. The existing hydro-generation equipment supplies approximately 54% of the 5500 kW required by the Fries plant. With the addition of the new facility, the plant will generate approximately 74% of its total electrical requirements. This demonstrates the viability of utilizing hydro-generation in the operation of an industrial facility. The project annually generates a National Energy Savings of 19,387 barrels of oil equivalent. The project was separated into four phases which lasted a total of 36 months and included construction, erection and demonstration.

  15. Ecological studies related to construction of the Defense Waste Processing Facility on the Savannah River Plant. FY 1983-84 annual report

    SciTech Connect

    Pechmann, J.H.K.; Semlitsch, R.D.; Lew, R.M.; Mayack, D.T.

    1984-12-01

    This report details the ecological studies, conducted during Fiscal Years FY- 1983 and 1984 by the Savannah River Ecology Laboratory (SREL), that relate to the construction of the Defense Waste Processing Facility (DWPF) on the Savannah River Plant (SRP) near Aiken, South Carolina. SREL has been contracted to carry out these studies for the Department of Energy (DOE) in order to provide data needed to show compliance with the National Environmental Policy Act (NEPA) of 1969, the Endangered Species Act of 1973, and Executive Order 11990 (Protection of Wetlands, 1977). Studies in previous years were directed toward compiling a biological inventory of both the DWPF construction site and alternative sites. This information was then utilized to predict the effects of construction on resident biota in the effected areas. Since the start of this construction of FY-1984, the focus of our efforts has shifted to verification of the predicted impacts, and evaluation of the effectiveness of mitigation measures. Particular emphasis is being placed on monitoring the effects of runoff from the site on peripheral streams in the SRP's Upper Three Runs watershed, and on artificial refuge ponds. These ponds, completed during FY-1983, represent an experimental attempt to mitigate the loss of an important amphibian breeding site, Sun Bay, to DWPF construction. Monitoring of a similar undisturbed wetland, Rainbow Bay, has continued in order to provide a comparable control site for these studies. 70 refs., 17 figs., 20 tabs.

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

    PubMed Central

    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

  17. The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

    PubMed Central

    Hou, Fajian; Chu, Chih-Wen; Kong, Xiangduo; Yokomori, Kyoko; Zou, Hui

    2007-01-01

    Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis. PMID:17502424

  18. Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani

    PubMed Central

    Yadav, Aarti; Chandra, Udita; Saha, Swati

    2016-01-01

    Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area. PMID:27272906

  19. Polymorphisms in the Human Cytochrome P450 and Arylamine N-Acetyltransferase: Susceptibility to Head and Neck Cancers

    PubMed Central

    Khlifi, Rim; Messaoud, Olfa; Rebai, Ahmed; Hamza-Chaffai, Amel

    2013-01-01

    The occurrence of head and neck cancer (HNC) is associated with smoking and alcohol drinking. Tobacco smoking exposes smokers to a series of carcinogenic chemicals. Cytochrome P450 enzymes (CYP450s), such as CYP1A1, CYP1B1, and CYP2D6, usually metabolize carcinogens to their inactive derivatives, but they occasionally convert the chemicals to more potent carcinogens. In addition, via CYP450 (CYP2E1) oxidase, alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Furthermore, two N-acetyltransferase isozymes (NATs), NAT1 and NAT2, are polymorphic and catalyze both N-acetylation and O-acetylation of aromatic and heterocyclic amine carcinogens. Genetic polymorphisms are associated with a number of enzymes involved in the metabolism of carcinogens important in the induction of HNC. It has been suggested that such polymorphisms may be linked to cancer susceptibility. In this paper, we select four cytochrome P450 enzymes (CYP1A1, CYP1BA1, CYP2D6, and CYP2E1), and two N-acetyltransferase isozymes (NAT1 and NAT2) in order to summarize and analyze findings from the literature related to HNC risk by focusing on (i) the interaction between these genes and the environment, (ii) the impact of genetic defect on protein activity and/or expression, and (iii) the eventual involvement of race in such associations. PMID:24151610

  20. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  1. The Polyamine N-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

    PubMed Central

    Martini, Cecilia; Michaux, Charlotte; Bugli, Francesca; Arcovito, Alessandro; Iavarone, Federica; Cacaci, Margherita; Sterbini, Francesco Paroni; Hartke, Axel; Sauvageot, Nicolas; Sanguinetti, Maurizio; Posteraro, Brunella

    2014-01-01

    We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N1-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism. PMID:25385793

  2. The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): Structure, chromosomal localization, and tissue expression

    SciTech Connect

    Coon, S.L.; Bernard, M.; Roseboom, P.H.

    1996-05-15

    Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, HGMW-approved symbol AANAT;EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and controls the night/day rhythm in melatonin production in the vertebrate pineal gland. We have found that the human AA-NAT gene spans {approx}2.5 kb, contains four exons, and is located at chromosome 17q25. The open reading frame encodes a 23.2-kDa protein that is {approx}80% identical to sheep and rat AA-NAT. The AA-NAT transcript ({approx}1 kb) is highly abundant in the pineal gland and is expressed at lower levels in the retina and in the Y79 retinoblastoma cell line. AA-NAT mRNA is also detectable at low levels in several brain regions and the pituitary gland, but not in several peripheral tissues examined. Brain and pituitary AA-NAT could modulate serotonin-dependent aspects of human behavior and pituitary function. 31 refs., 5 figs.

  3. Generation IV Nuclear Energy Systems Construction Cost Reductions through the use of Virtual Environments: Task 1 Completion Report

    SciTech Connect

    Whisker, V.E.; Baratta, A.J.; Shaw, T.S.; Winters, J.W.; Trikouros, N.; Hess, C.

    2002-11-26

    OAK B204 The objective of this project is to demonstrate the feasibility and effectiveness of using full-scale virtual reality simulation in the design, construction, and maintenance of future nuclear power plants. Specifically, this project will test the suitability of Immersive Projection Display (IPD) technology to aid engineers in the design of the next generation nuclear power plant and to evaluate potential cost reductions that can be realized by optimization of installation and construction sequences. The intent is to see if this type of information technology can be used in capacities similar to those currently filled by full-scale physical mockups.

  4. Report of independent consultants reviewing Integrated Test Stands (ITS) performance and readiness of DARHT for construction start

    SciTech Connect

    Not Available

    1993-08-01

    Independent consultants met at Los Alamos, June 15 and 16, 1993, to review progress on the commissioning of the Integrated Test Stand (ITS) for DARHT and to provide DOE with technical input on readiness for construction of the first radiographic arm of DARHT. The consultants concluded that all milestones necessary for demonstrating the performance of the DARHT accelerator have been met and that the project is ready for construction to resume. The experimental program using ITS should be continued to quantify the comparison of experiment and theory, to test improvements on the injector insulator, and to better evaluate the interaction of the beam and the target.

  5. Cost and schedule estimate to construct the tunnel and shaft remedial shielding concept, Los Alamos Meson Physics Facility, Los Alamos National Laboratory, Los Alamos, New Mexico. Final report

    SciTech Connect

    1993-11-30

    The report provides an estimate of the cost and associated schedule to construct the tunnel and shaft remedial shielding concept. The cost and schedule estimate is based on a preliminary concept intended to address the potential radiation effects on Line D and Line Facilities in event of a beam spill. The construction approach utilizes careful tunneling methods based on available excavation and ground support technology. The tunneling rates and overall productivity on which the cost and project schedule are estimated are based on conservative assumptions with appropriate contingencies to address the uncertainty associated with geological conditions. The report is intended to provide supplemental information which will assist in assessing the feasibility of the tunnel and shaft concept and justification for future development of this particular aspect of remedial shielding for Line D and Line D Facilities.

  6. Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair.

    PubMed

    Qi, Wenjing; Chen, Hongyu; Xiao, Ting; Wang, Ruoxi; Li, Ting; Han, Liping; Zeng, Xianlu

    2016-03-01

    Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.

  7. Report to the Board of Regents State University System of Florida. Review of Programs: Architecture, Architectural Technology, Landscape Architecture, Interior Design, Construction and Construction Technology, Building Construction, Urban and Regional Planning.

    ERIC Educational Resources Information Center

    McMinn, William G.

    An evaluation and report was done on the status of programs in architecture and related fields in the Florida State University System as a follow-up to a 1983 evaluation. The evaluation involved self-studies prepared by each program and a series of site visits to each of seven campuses and two centers with programs under review. These institutions…

  8. Construction and Analysis of Semantic Properties of an Alternate Form of the Personality Worksample. Technical Report 1990-6.

    ERIC Educational Resources Information Center

    Veccia, Ellen M.; Schroeder, David H.

    A set of 150 experimental personality items was constructed for an alternate form of the word association personality worksample developed by the Johnson O'Connor Research Foundation. The items were intended to possess several semantic properties hypothesized to facilitate discrimination between objective and subjective examinees. Specifically,…

  9. Construct Validity of a Set of Mathematical Superitems. A Report on the NIE/ECS Item Development Project.

    ERIC Educational Resources Information Center

    Romberg, Thomas A.; And Others

    Procedural documentation is presented for administering, scoring and analyzing data gathered to examine the construct validity of a set of superitems developed to assess student levels of mathematical reasoning ability. Each superitem includes a mathematical situation and a structured set of questions about that situation. The questions were based…

  10. Resume and Report of Visitations to Schools Employing the Open-Space Concept of School Construction ("Schools-Without-Walls").

    ERIC Educational Resources Information Center

    Ramsey, Robert

    Four schools were visited in an attempt to assess the merits of combining several classrooms in a large space as a method of school construction. Interviews with students and teachers as well as personal impressions form the basis for evaluation. Satisfactory results were found for schools-without-walls, school carpeting, acoustic control, team…

  11. Construct Validity of "e-rater"® in Scoring TOEFL® Essays. Research Report. ETS RR-07-21

    ERIC Educational Resources Information Center

    Attali, Yigal

    2007-01-01

    This study examined the construct validity of the "e-rater"® automated essay scoring engine as an alternative to human scoring in the context of TOEFL® essay writing. Analyses were based on a sample of students who repeated the TOEFL within a short time period. Two "e-rater" scores were investigated in this study, the first…

  12. Ecological studies related to construction of the Defense Waste Processing Facility on the Savannah River Site. Annual report, FY 1993

    SciTech Connect

    1994-11-01

    Construction of the Defense Waste Processing Facility (DWPF) on the Savannah River Site (SRS) began during FY-1984. The Savannah River Ecology Laboratory (SREL) has completed 15 years of ecological studies related to the construction of the DWPF complex. Prior to construction, the 600-acre site (S-Area) contained a Carolina bay and the headwaters of a stream. Through the long-term census taking of biota at the DWPF site and Rainbow Bay, SREL has been evaluating the impact of construction on the biota and the effectiveness of mitigation efforts. similarly, the effects of erosion from the DWPF site on the water quality of S-Area peripheral streams are being assessed. This research provides supporting data relevant to the National Environmental Policy Act (NEPA) of 1969, the Endangered Species Act of 1973, Executive orders 11988 (Floodplain Management) and 11990 (Protection of Wetlands), and United States Department of Energy (DOE) Guidelines for Compliance with Floodplain/Wetland Environmental Review Requirements (10 CFR 1022).

  13. The Development of a Theoretical Construct of the Concepts of Touch as They Relate to Nursing. Final Report.

    ERIC Educational Resources Information Center

    Barnett, Kathryn E.

    To develop a theoretical construct of touch as it relates to nursing, an extensive was made of the basic and accepted theories of touch, and a survey was conducted of current practices of touch by approximately 900 health personnel in two Dallas, Texas hospitals. Survey data were gathered by observation of 180 sessions involving approximately 540…

  14. 40 CFR 60.1375 - What reports must I submit before I submit my notice of construction?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY SOURCES Standards of Performance for Small Municipal Waste Combustion Units for Which Construction is Commenced... separation plan, as specified in § 60.1065. (2) Your revised materials separation plan, as specified in §...

  15. Innovation in the Management of Primary School Construction in Afghanistan. A Case Study. Educational Building Report 9.

    ERIC Educational Resources Information Center

    Ahmad, Fazel

    By 1973 there were very great disparities between the opportunities for education in the urban and rural areas of Afghanistan. This case study concerns provincial school construction programs for hundreds of small buildings in the remotest areas of what is one of the most mountainous countries of the world. A study proposed alternative building…

  16. Constructed-Response DIF Evaluations for Mixed-Format Tests. Research Report. ETS RR-13-33

    ERIC Educational Resources Information Center

    Moses, Tim; Liu, Jinghua; Tan, Adele; Deng, Weiling; Dorans, Neil J.

    2013-01-01

    In this study, differential item functioning (DIF) methods utilizing 14 different matching variables were applied to assess DIF in the constructed-response (CR) items from 6 forms of 3 mixed-format tests. Results suggested that the methods might produce distinct patterns of DIF results for different tests and testing programs, in that the DIF…

  17. Evaluation of the biotic potential of microorganisms and higher plants to enhance the quality of constructed wetlands. Final report

    SciTech Connect

    Mays, D.A.; Floyd, M.; Taylor, R.W.; Sistani, K.

    1998-09-30

    A project was carried out from October 1, 1991 through September 30, 1998 to evaluate the growth of several species of wetland plants in constructed cells using mine spoil as a growth medium, to evaluate microbial diversity and finally, to demonstrate the concept on an actual strip-mined site. In order to gain background information for evaluation of constructed wetlands, several wetlands on both undisturbed and strip-mined areas were evaluated to determine the physical and chemical characteristics of the substrates as well as the vegetation characteristics. The research phase of this projects consisted of 10 wetland cells each 7x16 m in size with the water depth varying from 0 to 40 cm. The substrates were allowed to stabilize over winter and each cell was planted in the spring of 1993 with 18 plants each of cattail, maidencance, soft stem bulrush and pickerel weed. All cells were thickly vegetated by the end of the first growing season.

  18. Evaluation and utilization of Illinois FBC residues for construction materials. [Quarterly] technical report, December 1, 1991--February 29, 1992

    SciTech Connect

    Ghafoori, N.; Sami, S.

    1992-08-01

    The overall objective of this program is to investigate the extent to which fluidized bed combustion (FBC) by-products can be properly utilized as the viable construction materials. This investigation focuses primarily on the properties of residues derived from fluidized combustion burning of Illinois high-sulfur coal. The research plan calls for evaluation of physico-chemical and engineering characteristics of the FBC-based cement and non-cement mixes. The results of this study will be used to compare the physical mechanical properties of the FBC-based mixtures with those of conventional mixes. The suitabililty of using FBC residues as a filler or binder aggregate for construction applications such as structural concrete members, precast building products, and as based or surface course for gravity dams and pavements in the form of conventional and roller compacted materials will then be evaluated.

  19. Utility residential new construction programs: Going beyond the code. A report from the Database on Energy Efficiency Programs (DEEP) Project

    SciTech Connect

    Vine, E.

    1995-08-01

    Based on an evaluation of 10 residential new construction programs, primarily sponsored by investor-owned utilities in the United States, we find that many of these programs are in dire straits and are in danger of being discontinued because current inclusion of only direct program effects leads to the conclusion that they are not cost-effective. We believe that the cost-effectiveness of residential new construction programs can be improved by: (1) promoting technologies and advanced building design practices that significantly exceed state and federal standards; (2) reducing program marketing costs and developing more effective marketing strategies; (3) recognizing the role of these programs in increasing compliance with existing state building codes; and (4) allowing utilities to obtain an ``energy-savings credit`` from utility regulators for program spillover (market transformation) impacts. Utilities can also leverage their resources in seizing these opportunities by forming strong and trusting partnerships with the building community and with local and state government.

  20. Office of Inspector General report on audit of renovation and new construction projects at Lawrence Livermore National Laboratory

    SciTech Connect

    1997-06-05

    The Oakland Operations Office (Oakland) is responsible for acquiring facilities needed to satisfy mission needs and to do so at the least cost to the Department of Energy (Department). The objective of the audit was to determine if proposed renovation and new construction projects at the Lawrence Livermore National Laboratory (Livermore) met mission needs while minimizing cost to the Government. In pursuing three projects, estimated to cost over $78 million, Livermore had not demonstrated that it had selected the best alternatives for meeting the Department`s needs while minimizing cost. Livermore was able to pursue these projects because Oakland did not ensure that the laboratory had performed cost and benefit analyses of all alternatives. Further, Oakland did not establish benchmarks to assess the reasonableness of the total costs of designing, constructing, and managing these projects. As a result, it was likely that the Department was spending more than necessary on renovation and new construction projects at Livermore. Although the projects met mission needs, it was recommended that the Manager, Oakland: (1) require Livermore to perform analyses of expected costs and benefits for alternatives; (2) evaluate the adequacy of Livermore`s cost and benefit analyses of alternatives; (3) establish benchmarks based on industry and other government agency cost data to assess the reasonableness of Livermore`s total design, construction, and project management costs; and (4) select the alternative that meets established needs at the least cost to the Government. Oakland agreed with the recommendations and will implement them starting with the Fiscal Year 1999 project submission and validation.

  1. A preliminary report on the use of cane and bamboo as basic construction materials for orthotic and prosthetic appliances.

    PubMed

    Banerji, B; Banerji, J B

    1984-08-01

    Cane and bamboo have been found to be viable and alternative basic construction materials for orthotic/prosthetic appliances and rehabilitation aids. Amongst the appliances made, the night splint and the upper limb splints have proved effective in field trials. The lower limb orthoses and prosthesis however are still at an experimental stage. Of the rehabilitation aids, walkers, crutches and wheelchairs have been found to be remarkably useful, cheap and light.

  2. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase.

    PubMed

    He, Yuan; Roth, Christian; Turkenburg, Johan P; Davies, Gideon J

    2014-01-01

    The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP_05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  3. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained by using the similar pat gene as a selectable marker. The latter cultivars ca...

  4. Bioprospecting for Trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes that vary in their Aaility to modify the mycotoxin deoxynivalenol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to mod...

  5. (US low-level radioactive waste management facility design, construction, and operation): Foreign trip report, July 22--30, 1989

    SciTech Connect

    Van Hoesen, S.D.; Bolinsky, J.

    1989-08-02

    The Martin Marietta Energy Systems, Inc., Team, consisting of representatives of the Engineering Division and Oak Ridge National Laboratory (ORNL), participated in a technology exchange program on French and US low-level radioactive waste (LLW) management facility design, construction, and operation. Meetings were held at the Agence National pour la Gestion des Dechets Radioactif (ANDRA) offices in Paris to review the designs for the new French LLW disposal facility, the Cente de Stockage de l'Aube (CSA), and the new ORNL LLW disposal project, the Interim Waste Management Facility (IWMF), and the results of the French LLW disposal facility cover experiment at St. Sauveur. Visits were made to the operating LLW disposal facility, the Centre de Stockage de la Manche (CSM), the LLW conditioning facilities at the La Hague Reprocessing Facility, and the St. Saueveur Disposal Cap Experiment to discuss design, construction, and operating experience. A visit was also made to the CSA site to view the progress made in construction of the new facility.

  6. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-05-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria.

  7. Closure Report for Corrective Action Unit 425: Area 9 Main Lake Construction Debris Disposal Area, Tonopah Test Range, Nevada

    SciTech Connect

    K. B. Campbell

    2003-03-01

    Corrective Action Unit (CAU) 425 is located on the Tonopah Test Range, approximately 386 kilometers (240 miles) northwest of Las Vegas, Nevada. CAU 425 is listed in the Federal Facility Agreement and Consent Order (FFACO, 1996) and is comprised of one Corrective Action Site (CAS). CAS 09-08-001-TA09 consisted of a large pile of concrete rubble from the original Hard Target and construction debris associated with the Tornado Rocket Sled Tests. CAU 425 was closed in accordance with the FFACO and the Nevada Division of Environmental Protection-approved Streamlined Approach for Environmental Restoration Plan for CAU 425: Area 9 Main Lake Construction Debris Disposal Area, Tonopah Test Range, Nevada (U.S. Department of Energy, Nevada Operations Office, 2002). CAU 425 was closed by implementing the following corrective actions: The approved corrective action for this unit was clean closure. Closure activities included: (1) Removal of all the debris from the site. (2) Weighing each load of debris leaving the job site. (3) Transporting the debris to the U.S. Air Force Construction Landfill for disposal. (4) Placing the radioactive material in a U.S. Department of Transportation approved container for proper transport and disposal. (5) Transporting the radioactive material to the Nevada Test Site for disposal. (6) Regrading the job site to its approximate original contours/elevation.

  8. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R.

    2013-01-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria. PMID:23353051

  9. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    SciTech Connect

    Thoden, James B.; Holden, Hazel M.

    2010-09-08

    The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

  10. The narrow active-site cleft of O-acetylserine sulfhydrylase from Leishmania donovani allows complex formation with serine acetyltransferases with a range of C-terminal sequences.

    PubMed

    Raj, Isha; Kumar, Sudhir; Gourinath, Samudrala

    2012-08-01

    Cysteine is a crucial substrate for the synthesis of glutathione and trypanothione, which in turn maintain intracellular redox homeostasis and defend against oxidative stress in the pathogen Leishmania donovani. Here, the identification, sequencing, characterization and crystal structure at 1.79 Å resolution of O-acetylserine sulfhydrylase (OASS), a cysteine-biosynthetic pathway enzyme from L. donovani (LdOASS), are reported. It shows binding to the serine acetyltransferase (SAT) C-terminal peptide, indicating that OASS and SAT interact with each other to form a cysteine synthase complex, further confirmed by the structure of LdOASS in complex with SAT C-terminal octapeptide at 1.68 Å resolution. Docking and fluorescence binding studies show that almost all SAT C-terminus mimicking tetrapeptides can bind to LdOASS. Some peptides had a higher binding affinity than the native peptide, indicating that SAT-OASS interactions are not sequence-specific. The structure of LdOASS with a designed peptide (DWSI) revealed that LdOASS makes more interactions with the designed peptide than with the native peptide. In almost all known SAT-OASS interactions the SAT C-terminal sequence was shown to contain amino acids with large side chains. Structural comparison with other OASSs revealed that LdOASS has a relatively less open active-site cleft, which may be responsible for its interaction with the smaller-amino-acid-containing C-terminal LdSAT peptide. Biochemical studies confirmed that LdOASS interacts with SATs from Entamoeba histolytica and Brucella abortus, further displaying its sequence-independent and versatile mode of interaction with SATs. This implicates a critical role of the size of the active-site cleft opening in OASS for SAT-OASS interaction and thus cysteine synthase complex formation.

  11. Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)

    PubMed Central

    Haer-Wigman, Lonneke; Newman, Hadas; Leibu, Rina; Bax, Nathalie M.; Baris, Hagit N; Rizel, Leah; Banin, Eyal; Massarweh, Amir; Roosing, Susanne; Lefeber, Dirk J.; Zonneveld-Vrieling, Marijke N.; Isakov, Ofer; Shomron, Noam; Sharon, Dror; Den Hollander, Anneke I.; Hoyng, Carel B.; Cremers, Frans P.M.; Ben-Yosef, Tamar

    2015-01-01

    Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina. PMID:25859010

  12. Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

    PubMed

    Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

    2015-10-22

    One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

  13. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; Burroughs, A. Maxwell; Aravind, L.

    2011-01-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family. PMID:20023723

  14. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Maxwell Burroughs, A; Aravind, L

    2009-12-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN-hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family.

  15. Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme*

    PubMed Central

    Frase, Hilary; Toth, Marta; Vakulenko, Sergei B.

    2012-01-01

    The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (kcat/Km = 105-107 m−1 s−1). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides. PMID:23115238

  16. 15-Deoxy-{Delta}{sup 12,14}-prostaglandin J{sub 2} impairs the functions of histone acetyltransferases through their insolubilization in cells

    SciTech Connect

    Hironaka, Asako; Morisugi, Toshiaki; Kawakami, Tetsuji; Miyagi, Ikuko; Tanaka, Yasuharu

    2009-12-11

    The cyclopentenonic prostaglandin 15-deoxy-{Delta}{sup 12,14}-PG J{sub 2} (15d-PGJ{sub 2}) is a metabolite derived from PGD{sub 2}. Although 15d-PGJ{sub 2} has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor {gamma} (PPAR{gamma}), the functions are not fully understood. In order to examine the effect of 15d-PGJ{sub 2} on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ{sub 2}. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-{kappa}B-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ{sub 2} attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ{sub 2} on the function of HATs was independent of PPAR{gamma}, because PPAR{gamma} agonists could not mimick 15d-PGJ{sub 2} and PPAR{gamma} antagonists did not inhibit 15d-PGJ{sub 2}.

  17. Parathyroid hormone activation of matrix metalloproteinase-13 transcription requires the histone acetyltransferase activity of p300 and PCAF and p300-dependent acetylation of PCAF.

    PubMed

    Lee, Minnkyong; Partridge, Nicola C

    2010-12-03

    Parathyroid hormone (PTH) regulates the transcription of many genes involved in bone remodeling in osteoblasts. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. We have previously shown that Mmp-13 gene expression is highly induced by PTH treatment in osteoblastic UMR 106-01 cells, as well as primary osteoblasts. Here, we show that p300/CBP-associated factor (PCAF), in addition to p300 and Runx2, is required for PTH activation of Mmp-13 transcription. PCAF was increasingly recruited to the MMP-13 proximal promoter region after PTH treatment, and this was associated with an increase in RNA polymerase II recruitment and histone acetylation. In addition, PTH treatment increased the acetylation of PCAF, a process that required p300. Knockdown of PCAF, p300, or Runx2 by siRNA decreased Mmp-13 mRNA expression after PTH treatment in both UMR 106-01 cells and primary osteoblasts. We found that there is a mutual dependence between p300 and PCAF to be recruited to the Mmp-13 promoter after PTH treatment. In promoter-reporter assays, p300 and PCAF had an additive effect on PTH stimulation of MMP-13 promoter activity, and this required their histone acetyltransferase activity. Our findings demonstrate that PCAF acts downstream of PTH signaling as a transcriptional coactivator that is required for PTH stimulation of MMP-13 transcription. PCAF cooperates with p300 and Runx2 to mediate PTH activation of MMP-13 transcription.

  18. Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype*

    PubMed Central

    Purushothaman, Anurag; Hurst, Douglas R.; Pisano, Claudio; Mizumoto, Shuji; Sugahara, Kazuyuki; Sanderson, Ralph D.

    2011-01-01

    Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype. PMID:21757697

  19. Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

    SciTech Connect

    Phelps, P.E.; Barber, R.P.; Vaughn, J.E.

    1988-07-22

    This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord.

  20. Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

    PubMed Central

    Mogollon, Catherin Marin; van Pul, Fiona J. A.; Imai, Takashi; Ramesar, Jai; Chevalley-Maurel, Séverine; de Roo, Guido M.; Veld, Sabrina A. J.; Kroeze, Hans; Franke-Fayard, Blandine M. D.; Janse, Chris J.

    2016-01-01

    The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters. PMID:27997583