Table 4. Essential Genes
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METACYC ID Description A0AR23 GO:0004842 (Ubiquitin-Protein Ligase
Electronic Supplementary Material (ESI) for Integrative Biology This journal is © The Royal Society of Chemistry 2012 Heat Stress Responsive Zostera marina Genes, Southern Population (α=0. -
I HIGH MASS ACCURACY COUPLED to SPATIALLY-DIRECTED
HIGH MASS ACCURACY COUPLED TO SPATIALLY-DIRECTED PROTEOMICS FOR IMPROVED PROTEIN IDENTIFICATIONS IN IMAGING MASS SPECTROMETRY EXPERIMENTS By David Geoffrey Rizzo Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry August, 2016 Nashville, Tennessee Approved: Richard M. Caprioli, Ph.D. Kevin L. Schey, Ph.D. John A. McLean, Ph.D. Michael P. Stone, Ph.D. i Copyright © 2016 by David Geoffrey Rizzo All Rights Reserved ii This work is dedicated to my family and friends, who have shown nothing but support for me in all of life’s endeavors. iii ACKNOWLEDGEMENTS “As we express our gratitude, we must never forget that the highest appreciation is not to utter words, but to live by them.” - John F. Kennedy – There are many people I must thank for showing kindness, encouragement, and support for me during my tenure as a graduate student. First and foremost, I would like to thank my research advisor, Richard Caprioli, for providing both ample resources and guidance that allowed me to grow as a scientist. Our discussions about my research and science in general have helped me become a much more focused and discerning analytical chemist. I must also thank my Ph.D. committee members, Drs. Kevin Schey, John McLean, and Michael Stone, who have brought valuable insight into my research and provided direction along the way. My undergraduate advisor, Dr. Facundo Fernández, encouraged me to begin research in his lab and introduced me to the world of mass spectrometry. -
In the Field of Clinical Examination, the Measurement of Creatine
J. Clin. Biochem. Nutr., 3, 17-25, 1987 Bacterial Glucokinase as an Enzymic Reagent of Good Stability for Measurement of Creatine Kinase Activity Hitoshi KONDO, * Takanari SHIRAISHI, Masao KAGEYAMA, Kazuhiko NAGATA, and Kosuke TOMITA Research and Development Center, UNITIKA Ltd., Uji 611, Japan (Received January 10, 1987) Summary An enzymic reagent, that has long-term stability even in the liquid state, was successfully employed for the measurement of serum creatine kinase (CK, EC 2.7.3.2) activity. The enzyme used was the thermostable glucokinase (GlcK, EC 2.7.1.2) obtained from the thermo- phile Bacillus stearothermophilus. The reagent was found to be stable in solution for about one month at 6•Ž and for about one week at 30•Ž. This substitution of glucokinase for the hexokinase of the most commonly used hexokinase-glucose-6-phosphate dehydrogenase (HK-G6PDH) method results in a remarkable improvement of the method. The CK activity measured by the GlcK-G6PDH method was linear up to about 2,000 U/ liter at 37•Ž. The GlcK-G6PDH method was found to give a satisfactory precision and reproducibility (coefficient of variation less than 2.17%). Over a wide range of CK activity, an excellent agreement was obtained between the GlcK-G6PDH and the HK-G6PDH methods. Furthermore several coexistents and anticoagulants were found to have little effect on the measured value of CK activity by the GlcK-G6PDH method. Key Words: creatine kinase activity, glucokinase, improved stability of reagent, creatine kinase determination, thermostable enzyme In the field of clinical examination, the measurement of creatine kinase (CK, ATP : creatine phosphotransferase, EC 2.7.3.2) activity in serum is one of the important examinations usually employed for diagnosis of cardiac diseases such as myocardial infarction or muscular diseases such as progressive muscular dystrophy. -
Phosphatidylinositol-3-Kinase in Tomato (Solanum Lycopersicum. L) Fruit and Its Role in Ethylene Signal Transduction and Senescence
Phosphatidylinositol-3-Kinase in Tomato (Solanum lycopersicum. L) Fruit and Its Role in Ethylene Signal Transduction and Senescence by Mohd Sabri Pak Dek A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Plant Agriculture Guelph, Ontario, Canada © Mohd Sabri Pak Dek,June, 2015 ABSTRACT PHOSPHATIDYLINOSITOL-3-KINASE IN TOMATO (SOLANUM LYCOPERSICUM. L) FRUIT AND ITS ROLE IN ETHYLENE SIGNAL TRANSDUCTION AND SENESCENCE Mohd Sabri Pak Dek Co-Advisors: University of Guelph, 2015 Professor G. Paliyath Professor J. Subramanian The ripening process is initiated by ethylene through a signal transduction cascade leads to the expression of ripening-related genes and catabolism of membrane, cell wall, and storage components. One of the minor components in membrane phospholipids is phosphatidylinositol (PI). Phosphatidylinositol-3-kinase (PIK) is an enzyme that phosphorylates PI at the 3-OH position of inositol head group to produce phosphatidylinositol 3-phosphate (PI3P). Phosphorylation of PI may be an early event in the ethylene signal transduction pathway that generates negatively charged domains on the plasma membrane. PI3P domains may potentially serve as a docking site for phospholipase D (PLD) after ethylene stimulation. It is hypothesized that ethylene stimulation may activate PI3K resulting in enhanced level of phosphorylated phosphatidylinositol. However, the properties and function of PI3K is not well understood in plants. In the present study, the effect of PI3K inhibition during tomato fruit ripening was evaluated. This study demonstrated that PI3K activity is required for normal ripening process of the fruit. Inhibition of PI3K activity using wortmannin significantly reduced tomato ripening process. -
Non-Homologous Isofunctional Enzymes: a Systematic Analysis Of
Omelchenko et al. Biology Direct 2010, 5:31 http://www.biology-direct.com/content/5/1/31 RESEARCH Open Access Non-homologousResearch isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution Marina V Omelchenko, Michael Y Galperin*, Yuri I Wolf and Eugene V Koonin Abstract Background: Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. Results: We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. -
Labeled in Thecourse of Glycolysis, Since Phosphoglycerate Kinase
THE STATE OF MAGNESIUM IN CELLS AS ESTIMATED FROM THE ADENYLATE KINASE EQUILIBRIUM* BY TRWIN A. RoSE THE INSTITUTE FOR CANCER RESEARCH, PHILADELPHIA Communicated by Thomas F. Anderson, August 30, 1968 Magnesium functions in many enzymatic reactions as a cofactor and in com- plex with nucleotides acting as substrates. Numerous examples of a possible regulatory role of Mg can be cited from studies with isolated enzymes,'- and it is known that Mg affects the structural integrity of macromolecules such as trans- fer RNA" and functional elements such as ribosomes.'0 The major problem in translating this information on isolated preparations to the functioning cell is the difficulty in determining the distribution of Mg and the nucleotides among the free and complexed forms that function in the region of the cell for which this information is desired. Nanningall based an attempt to calculate the free Mg2+ and Ca2+ ion concentrations of frog muscle on the total content of these metals and of the principal known ligands (adenosine 5'-triphosphate (ATP), creatine-P, and myosin) and the dissociation constants of the complexes. However, this method suffers from the necessity of evaluating the contribution of all ligands as well as from the assumption that all the known ligands are contributing their full complexing capacity. During studies concerned with the control of glycolysis in red cells and the control of the phosphoglycerate kinase step in particular, it became important to determine the fractions of the cell's ATP and adenosine 5'-diphosphate (ADP) that were present as Mg complexes. Just as the problem of determining the distribution of protonated and dissociated forms of an acid can be solved from a knowledge of pH and pKa of the acid, so it would be possible to determine the liganded and free forms of all rapidly established Mg complexes from a knowledge of Mg2+ ion concentration and the appropriate dissociation constants. -
Guanylate Kinase (Ec 2.7.4.8)
Enzymatic Assay of GUANYLATE KINASE (EC 2.7.4.8) PRINCIPLE: Guanylate Kinase GMP + ATP > GDP + ADP Pyruvate Kinase ADP + PEP > ATP + Pyruvate Pyruvate Kinase GDP + PEP > GTP + Pyruvate Lactic Dehydrogenase 2 Pyruvate + 2 ß-NADH > 2 Lactate + 2 ß-NAD Abbreviations used: GMP = Guanosine 5'-Monophosphate ATP = Adenosine 5'-Triphosphate GDP = Guanosine 5'-Diphosphate ADP = Adenosine 5'-Diphosphate PEP = Phospho(enol)phosphate ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form CONDITIONS: T = 30°C, pH = 7.5, A340nm, Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 200 mM Tris HCl Buffer, pH 7.5 at 30°C (Prepare 50 ml in deionized water using Trizma Base, Sigma Prod. No. T-1503. Adjust to pH 7.5 at 30°C with 1 M HCl.) B. 1 M Potassium Chloride Solution (KCl) (Prepare 10 ml in deionized water using Potassium Chloride, Sigma Prod. No. P-4504.) C. 60 mM Magnesium Sulfate Solution (MgSO4) (Prepare 20 ml in deionized water using Magnesium Sulfate, Heptahydrate, Sigma Prod. No. M-1880.) D. 40 mM Phospho(enol)pyruvate Solution (PEP) (Prepare 50 ml in deionized water using Phospho(enol)Pyruvate, Trisodium Salt, Hydrate, Sigma Prod. No. P-7002. PREPARE FRESH.) Revised: 03/10/94 Page 1 of 4 Enzymatic Assay of GUANYLATE KINASE (EC 2.7.4.8) REAGENTS: (continued) E. 100 mM Ethylenediaminetetraacetic Acid Solution (EDTA) (Prepare 10 ml in deionized water using Ethylenediaminetetraacetic Acid, Tetrasodium Salt, Hydrate, Sigma Stock No. ED4SS.) F. 3.8 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form (ß-NADH) (Prepare 2 ml in deionized water using ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Dipotassium Salt, Sigma Prod. -
WO 2013/180584 Al 5 December 2013 (05.12.2013) P O P C T
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/180584 Al 5 December 2013 (05.12.2013) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 1/21 (2006.01) C12N 15/74 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 15/52 (2006.01) C12P 5/02 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C12N 15/63 (2006.01) HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/NZ20 13/000095 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, (22) International Filing Date: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 4 June 2013 (04.06.2013) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 61/654,412 1 June 2012 (01 .06.2012) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (71) Applicant: LANZATECH NEW ZEALAND LIMITED MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, [NZ/NZ]; 24 Balfour Road, Parnell, Auckland, 1052 (NZ). -
Polymerase Ribozyme with Promoter Recognition
In vitro Evolution of a Processive Clamping RNA Polymerase Ribozyme with Promoter Recognition by Razvan Cojocaru BSc, Simon Fraser University, 2014 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biochemistry Faculty of Science © Razvan Cojocaru 2021 SIMON FRASER UNIVERSITY Summer 2021 Copyright in this work is held by the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Declaration of Committee Name: Razvan Cojocaru Degree: Doctor of Philosophy Title: In vitro Evolution of a Processive Clamping RNA Polymerase Ribozyme with Promoter Recognition Committee: Chair: Lisa Craig Professor, Molecular Biology and Biochemistry Peter Unrau Supervisor Professor, Molecular Biology and Biochemistry Dipankar Sen Committee Member Professor, Molecular Biology and Biochemistry Michel Leroux Committee Member Professor, Molecular Biology and Biochemistry Mani Larijani Internal Examiner Associate Professor, Molecular Biology and Biochemistry Gerald Joyce External Examiner Professor, Jack H. Skirball Center for Chemical Biology and Proteomics Salk Institute for Biological Studies Date Defended/Approved: August 12, 2021 ii Abstract The RNA World hypothesis proposes that the early evolution of life began with RNAs that can serve both as carriers of genetic information and as catalysts. Later in evolution, these functions were gradually replaced by DNA and enzymatic proteins in cellular biology. I start by reviewing the naturally occurring catalytic RNAs, ribozymes, as they play many important roles in biology today. These ribozymes are central to protein synthesis and the regulation of gene expression, creating a landscape that strongly supports an early RNA World. -
Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile*
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 19, Issue of May 10, pp. 17334–17348, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile* Received for publication, October 23, 2001, and in revised form, February 18, 2002 Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M110198200 Irina Bruck‡, Alexander Yuzhakov§¶, Olga Yurieva§, David Jeruzalmi§, Maija Skangalis‡§, John Kuriyan‡§, and Mike O’Donnell‡§ʈ From §The Rockefeller University and ‡Howard Hughes Medical Institute, New York, New York 10021 This report takes a proteomic/genomic approach to polymerase III (pol III) structure and function has been ob- characterize the DNA polymerase III replication appa- tained from studies of the Escherichia coli replicase, DNA ratus of the extreme thermophile, Aquifex aeolicus. polymerase III holoenzyme (reviewed in Ref. 6). Therefore, a Genes (dnaX, holA, and holB) encoding the subunits re- brief overview of its structure and function is instructive for the ␦ ␦ quired for clamp loading activity ( , , and ) were iden- comparisons to be made in this report. In E. coli, the catalytic Downloaded from tified. The dnaX gene produces only the full-length subunit of DNA polymerase III is the ␣ subunit (129.9 kDa) product, , and therefore differs from Escherichia coli encoded by dnaE; it lacks a proofreading exonuclease (7). The dnaX that produces two proteins (␥ and ). Nonetheless, Ј Ј ⑀ ␦␦ proofreading 3 –5 -exonuclease activity is contained in the the A. aeolicus proteins form a complex. The dnaN ␣ ,␦␦ (27.5 kDa) subunit (dnaQ) that forms a 1:1 complex with (8  gene encoding the clamp was identified, and the ␣Ϫ⑀  9). -
Negative Regulation of Diacylglycerol Kinase &Theta
Cell Death and Differentiation (2010) 17, 1059–1068 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd Negative regulation of diacylglycerol kinase h mediates adenosine-dependent hepatocyte preconditioning G Baldanzi1,5, E Alchera2,5, C Imarisio2, M Gaggianesi1, C Dal Ponte2, M Nitti3, C Domenicotti3, WJ van Blitterswijk4, E Albano2, A Graziani1,5 and R Carini*,2,5 In liver ischemic preconditioning (IP), stimulation of adenosine A2a receptors (A2aR) prevents ischemia/reperfusion injury by promoting diacylglycerol-mediated activation of protein kinase C (PKC). By concerting diacylglycerol to phosphatidic acid, diacylglycerol kinases (DGKs) act as terminator of diacylglycerol signalling. This study investigates the role of DGK in the development of hepatocyte IP. DGK activity and cell viability were evaluated in isolated rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by the treatment with the A2aR agonist, CGS21680, and subsequently exposed to prolonged hypoxia. We observed that after IP or A2aR activation, a decrease in DGK activity was associated with the onset of hepatocyte tolerance to hypoxia. CGS21680-induced stimulation of A2aR specifically inhibited DGK isoform h by activating RhoA–GTPase. Consistently, both siRNA-mediated downregulation of DGK h and hepatocyte pretreatment with the DGK inhibitor R59949 induced cell tolerance to hypoxia. The pharmacological inhibition of DGK was associated with the diacylglycerol- dependent activation of PKC d and e and of their downstream target p38 MAPK. In conclusion, we unveil a novel signalling pathway contributing to the onset of hepatocyte preconditioning, which through RhoA–GTPase, couples A2aR to the downregulation of DGK. -
(12) Patent Application Publication (10) Pub. No.: US 2014/0155567 A1 Burk Et Al
US 2014O155567A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0155567 A1 Burk et al. (43) Pub. Date: Jun. 5, 2014 (54) MICROORGANISMS AND METHODS FOR (60) Provisional application No. 61/331,812, filed on May THE BIOSYNTHESIS OF BUTADENE 5, 2010. (71) Applicant: Genomatica, Inc., San Diego, CA (US) Publication Classification (72) Inventors: Mark J. Burk, San Diego, CA (US); (51) Int. Cl. Anthony P. Burgard, Bellefonte, PA CI2P 5/02 (2006.01) (US); Jun Sun, San Diego, CA (US); CSF 36/06 (2006.01) Robin E. Osterhout, San Diego, CA CD7C II/6 (2006.01) (US); Priti Pharkya, San Diego, CA (52) U.S. Cl. (US) CPC ................. CI2P5/026 (2013.01); C07C II/I6 (2013.01); C08F 136/06 (2013.01) (73) Assignee: Genomatica, Inc., San Diego, CA (US) USPC ... 526/335; 435/252.3:435/167; 435/254.2: (21) Appl. No.: 14/059,131 435/254.11: 435/252.33: 435/254.21:585/16 (22) Filed: Oct. 21, 2013 (57) ABSTRACT O O The invention provides non-naturally occurring microbial Related U.S. Application Data organisms having a butadiene pathway. The invention addi (63) Continuation of application No. 13/101,046, filed on tionally provides methods of using Such organisms to produce May 4, 2011, now Pat. No. 8,580,543. butadiene. Patent Application Publication Jun. 5, 2014 Sheet 1 of 4 US 2014/O155567 A1 ?ueudos!SMS |?un61– Patent Application Publication Jun. 5, 2014 Sheet 2 of 4 US 2014/O155567 A1 VOJ OO O Z?un61– Patent Application Publication US 2014/O155567 A1 {}}} Hººso Patent Application Publication Jun.