DOCSLIB.ORG

Multi-Omic Understanding of the Evolution of Xenobiotic Tolerance in Bacterial Isolates and Communities

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Multi-Omic Understanding of the Evolution of Xenobiotic Tolerance in Bacterial Isolates and Communities Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2019 Multi-omic Understanding of the Evolution of Xenobiotic Tolerance in Bacterial Isolates and Communities Tayte Paul Campbell Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Bioinformatics Commons, Biology Commons, and the Microbiology Commons Recommended Citation Campbell, Tayte Paul, "Multi-omic Understanding of the Evolution of Xenobiotic Tolerance in Bacterial Isolates and Communities" (2019). Arts & Sciences Electronic Theses and Dissertations. 1888. https://openscholarship.wustl.edu/art_sci_etds/1888 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Plant and Microbial Biosciences Dissertation Examination Committee: Gautam Dantas, Chair Arpita Bose Andrew Kau Audrey Odom-John Himadri Pakrasi Fuzhong Zhang Multi-omic Understanding of the Evolution of Xenobiotic Tolerance in Bacterial Isolates and Communities by Tayte P. Campbell A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2019 St. Louis, Missouri © 2019, Tayte P. Campbell Table of Contents List of Figures ................................................................................................................................ iv List of Tables ................................................................................................................................. iv Acknowledgments......................................................................................................................... vii Abstract .......................................................................................................................................... ix Chapter 1: Introduction ................................................................................................................... 1 1.1 Xenobiotics ........................................................................................................................... 1 1.2 Mechanisms of Xenobiotic Toxicity ..................................................................................... 2 1.3 Xenobiotic Tolerance ............................................................................................................ 3 1.4 Lignocellulose Degradation for Biofuel Production ............................................................. 4 1.5 Antibiotic Resistance in Gut Microbial Communities .......................................................... 6 Chapter 2: Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus ..................................................................................................... 7 2.1 Abstract ................................................................................................................................. 7 2.2 Introduction ........................................................................................................................... 8 2.3 Results ................................................................................................................................. 12 2.3.1 Adaptive evolution and characterization of adapted strains ........................................ 12 2.3.2 Comparative genomics of adapted strains ................................................................... 14 2.3.3 Characterization of adapted strains on lignin model compound mixtures ................... 16 2.3.4 Aromatic consumption in a lignin model compound mixture by PVHG6 .................. 17 2.3.5 Consumption of individual aromatic compounds by PVHG6 ..................................... 19 2.3.6 Transcriptomic analysis of WT and PVHG6 using lignin model compounds............. 19 2.3.7 Lipid production using lignin model compounds ........................................................ 29 2.4 Discussion ........................................................................................................................... 30 2.5 Materials and Methods ........................................................................................................ 33 2.5.1 Chemicals and strains .................................................................................................. 33 2.5.2 Adaptive evolution and growth assays of adapted strains ........................................... 34 2.5.3 R. opacus cultures for targeted metabolomics and transcriptomics ............................. 36 2.5.4 DNA extraction and sequencing library preparation ................................................... 37 2.5.5 Genome sequencing and SNP analysis ........................................................................ 37 2.5.6 RNA extraction and rRNA depletion ........................................................................... 38 2.5.7 Transcriptomic analysis ............................................................................................... 39 2.5.8 Aromatic consumption profiling .................................................................................. 39 2.5.9 Lipid assay ................................................................................................................... 40 2.5.10 Generation of R. opacus knockout mutants ............................................................... 41 2.5.10 Data Availability ........................................................................................................ 41 2.6 Supplemental Figures.......................................................................................................... 42 2.7 Acknowledgments............................................................................................................... 77 Chapter 3 ....................................................................................................................................... 78 ii 3.1 Abstract ............................................................................................................................... 78 3.2 Introduction ......................................................................................................................... 79 3.3 Results & Discussion .......................................................................................................... 82 3.3.1 Taxonomic composition of the gut microbiome was predominantly influenced by lifestyle .................................................................................................. 82 3.3.2 Functional pathways in the microbiome are relatively robust to changes in individual taxa.......................................................................................................... 86 3.3.3 Microbial resistome is primarily influenced by host lifestyle ..................................... 88 3.3.4 Functional metagenomic selections identified novel antibiotic resistance genes ........ 91 3.3.5 Captive ape microbiomes and resistomes cluster closely with non-Westernized humans ......................................................................................................................... 93 3.4 Materials and Methods ........................................................................................................ 98 3.4.1 Sample Collection ........................................................................................................ 98 3.4.2 DNA Extraction and Library Preparation .................................................................... 98 3.4.3 16S and shotgun metagenomics bioinformatics analysis............................................. 99 3.4.4 Functional metagenomics and analysis ...................................................................... 100 3.4.5 Microbiome and resistome comparisons to published cohorts .................................. 101 3.4.6 Colistin resistance analysis ........................................................................................ 102 3.4.7 Data availability ......................................................................................................... 102 3.5 Supplemental Figures........................................................................................................ 104 3.6 Acknowledgements ........................................................................................................... 111 Chapter 4 ..................................................................................................................................... 112 4.1 Lignocellulose degradation by R. opacus ......................................................................... 112 4.2 Antibiotic resistance in human and ape microbiomes ...................................................... 114 4.3 General Conclusion ........................................................................................................... 115 References ................................................................................................................................... 117 iii List
Load more

Recommended publications
  • Bacterial Oxidases of the Cytochrome Bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets
    Published in "Antioxidants & Redox Signaling doi: 10.1089/ars.2020.8039, 2020" which should be cited to refer to this work. Bacterial Oxidases of the Cytochrome bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets Vitaliy B. Borisov,1 Sergey A. Siletsky,1 Alessandro Paiardini,2 David Hoogewijs,3 Elena Forte,2 Alessandro Giuffre`,4 and Robert K. Poole5 Abstract Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria for- mally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, perox- ynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase.
    [Show full text]
  • In-Vitro Synthesis and Reconstitution of Cytochrome Bo3 Ubiquinol Oxidase in Artificial Membranes
    In-vitro Synthesis and Reconstitution of Cytochrome bo3 Ubiquinol Oxidase in Artificial Membranes Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universität Mainz Ahu ARSLAN YILDIZ geboren am 20.12.1980 in Turkey Mainz, 2010 Dekan: Prof. Dr. Erwin Schmidt 1. Berichterstatter: Prof. Dr. Eva K. Sinner 2. Berichterstatter: Prof. Dr. Harald Paulsen 3. Prüfer: Prof. Dr. Wolfgang Knoll 4. Prüfer: Prof. Dr. Elmar Jaenicke Tag der mündlichen Prüfung: 29.01.2010 Abbreviations ATP Adenosine triphosphate PD Parkinson’s Disease Cyt-bo3 Cytochrome bo3 ubiquinol oxidase NADH Nicotinamide adenine dinucleotide UQ Ubiquinone UQH2 Ubiquinol Cyt-c Cytochrome c E.coli Escherichia coli Heme Heme or Haem molecule Cyt-bd Cytochrome bd ubiquinol oxidase cyo Cytochrome encoding operon CuA Copper site in subunit II CuB Copper of binuclear site CFPS Cell-free protein synthesis tRNA Transfer RNA GTP Guanidine triphosphate BLMs Black lipid membranes sBLM Supported bilayer lipid membrane tBLM Tethered lipid bilayer membrane SPR Surface Plasmon Resonance Spectroscopy SPFS Surface Plasmon Enhanced Fluorescence Spectroscopy θc Critical angle θm Minimum angle TIR Total internal reflection εd Dielectric constant n Refractive index CA Contact angle pJRHisA Subunit II histidine tagged enzyme with natural promoter pRCO3 Fused subunit II-I-III enzyme with natural promoter pETcyo Subunit II histidine tagged enzyme with T7 promoter LB Luria Bertani media ii KOH Potassium hydroxide PES Polyethylene sulfonate
    [Show full text]
  • Polychlorinated Biphenyls in Pigments
    doi: 10.1111/cote.12167 Polychlorinated biphenyls in pigments: inadvertent production and environmental Coloration significance Technology Lisa Rodenburg,a Jia Guoa and Robert Christieb,* aDepartment of Environmental Science, Rutgers, The State University of New Jersey, New Brunswick, NJ, 08901, USA bSchool of Textiles and Design, Heriot-Watt University, Scottish Borders Campus, Netherdale, Galashiels, TD1 3HF, UK Feature article Email: [email protected] Society of Dyers and Colourists Received: 18 December 2014; Accepted: 26 June 2015 Polychlorobiphenyls are toxic, bioaccumulative, and persistent chemicals whose intentional manufacture has been banned throughout the developed world. Polychlorobiphenyls may be generated inadvertently during the production of certain pigments, including diarylides. This inadvertent production is allowed under various regulatory schemes, such as the Toxic Substances Control Act in the United States and the Stockholm Convention on Persistent Organic Pollutants. Generally, these regulations require polychlorobiphenyl levels in batches of pigment to be less than certain regulatory limits, usually 50 ppm. A growing body of evidence suggests that the use of pigments is dispersing polychlorobiphenyls throughout the environment. Polychlorobiphenyl congeners associated with pigments have been found throughout the United States in sediments and in surface waters at levels exceeding the prevailing water quality standards. A recent Japanese government study reported measured polychlorobiphenyl concentrations well
    [Show full text]
  • Metabolic and Regulatory Rearrangements Underlying Glycerol Metabolism in 3 Pseudomonas Putida KT2440 4 5 by 6 7 Pablo I
    1 1 2 Metabolic and regulatory rearrangements underlying glycerol metabolism in 3 Pseudomonas putida KT2440 4 5 by 6 7 Pablo I. Nikel, Juhyun Kim, and Víctor de Lorenzo* 8 9 Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 10 Madrid 28049, Spain 11 12 13 14 Running Title: Glycerol metabolism in P. putida 15 Keywords: Glycerol, Pseudomonas putida, central metabolism, Entner-Doudoroff pathway, redox 16 homeostasis, transcriptional regulation 17 18 19 20 _________________________________________________________________________________ 21 22 * Corresponding author V. de Lorenzo 23 Systems and Synthetic Biology Program 24 Centro Nacional de Biotecnología (CNB-CSIC) 25 Darwin 3, Campus de Cantoblanco 26 Madrid 28049, Spain 27 Phone: +34 91 585 4536 28 Fax: +34 91 585 4506 29 E-mail: [email protected] 30 2 1 SUMMARY 2 3 While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant 4 amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions 5 required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown 6 P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in 7 the shift between customary C sources (e.g., glucose or succinate) to the polyol. Transcriptomic results 8 were contrasted with key enzymatic activities under the same culture conditions. Cognate expression 9 profiles revealed that genes encoding enzymes of the Entner-Doudoroff route and other catabolic 10 pathways, e.g., the gluconate and 2-ketogluconate loops, were significantly down-regulated on glycerol.
    [Show full text]
  • Differential Effects of Epinephrine, Norepinephrine, and Indole
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M University INFECTION AND IMMUNITY, Sept. 2007, p. 4597–4607 Vol. 75, No. 9 0019-9567/07/$08.00ϩ0 doi:10.1128/IAI.00630-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Differential Effects of Epinephrine, Norepinephrine, and Indole on Escherichia coli O157:H7 Chemotaxis, Colonization, and Gene Expressionᰔ Tarun Bansal, Derek Englert, Jintae Lee, Manjunath Hegde, Thomas K. Wood, and Arul Jayaraman* Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843-3122 Received 3 May 2007/Returned for modification 4 June 2007/Accepted 13 June 2007 During infection in the gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) O157:H7 is exposed to a wide range of signaling molecules, including the eukaryotic hormones epinephrine and norepi- nephrine, and bacterial signal molecules such as indole. Since these signaling molecules have been shown to be involved in the regulation of phenotypes such as motility and virulence that are crucial for EHEC infections, we hypothesized that these molecules also govern the initial recognition of the large intestine environment and attachment to the host cell surface. Here, we report that, compared to indole, epinephrine and norepinephrine exert divergent effects on EHEC chemotaxis, motility, biofilm formation, gene expression, and colonization of HeLa cells. Using a novel two-fluorophore chemotaxis assay, it was found that EHEC is attracted to epineph- rine and norepinephrine while it is repelled by indole. In addition, epinephrine and norepinephrine also increased EHEC motility and biofilm formation while indole attenuated these phenotypes.
    [Show full text]
  • Davidge JBC Supplementary.Pdf
    promoting access to White Rose research papers Universities of Leeds, Sheffield and York http://eprints.whiterose.ac.uk/ White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/7923/ (includes links to Main Article, Supplementary Material and Figures) Published paper Davidge, K.S., Sanguinetti, G., Yee, C.H., Cox, A.G., McLeod, C.W., Monk, C.E., Mann, B.E., Motterlini, R. and Poole, R.K. (2009) Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators. Journal of Biological Chemistry, 284 (7). pp. 4516-4524. http://dx.doi.org/10.1074/jbc.M808210200 Supplementary Material White Rose Research Online [email protected] Supplementary Material Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators Kelly S Davidge, Guido Sanguinetti, Chu Hoi Yee, Alan G Cox, Cameron W McLeod, Claire E Monk, Brian E Mann, Roberto Motterlini and Robert K Poole Contents Page Number Supplementary Figure S1 3 Inhibition by CORM-3 of E. coli cultures grown in defined medium anaerobically and aerobically Supplementary Figure S2 4 Viability assays showing survival of anaerobically and aerobically E. coli in defined growth medium Supplementary Figure S3 5 Reaction of terminal oxidases in vivo on addition of RuCl2(DMSO)4 to intact cells in a dual-wavelength spectrophotometer Supplementary Figure S4 6 CORM-3 generates carbonmonoxycytochrome bd in vivo and depresses synthesis of cytochrome bo' Supplementary Figure S5 7 Expression of spy-lacZ activity
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • Development of a Transplantable Liver Graft from a Tiny Partial Liver( Dissertation 全文 )
    Auxiliary xenotransplantation as an in vivo bioreactor － Title Development of a transplantable liver graft from a tiny partial liver( Dissertation_全文 ) Author(s) Masano, Yuki Citation 京都大学 Issue Date 2020-03-23 URL https://doi.org/10.14989/doctor.r13329 Right https://onlinelibrary.wiley.com/journal/13993089 Type Thesis or Dissertation Textversion ETD Kyoto University Received: 27 November 2018 | Revised: 25 May 2019 | Accepted: 27 June 2019 DOI: 10.1111/xen.12545 ORIGINAL ARTICLE Auxiliary xenotransplantation as an in vivo bioreactor— Development of a transplantable liver graft from a tiny partial liver Yuki Masano1 | Shintaro Yagi1 | Yosuke Miyachi1 | Shinya Okumura1 | Toshimi Kaido1 | Hironori Haga2 | Eiji Kobayashi3 | Shinji Uemoto1 1Division of Hepato‐Biliary‐Pancreatic and Transplant Surgery, Department of Surgery, Abstract Graduate School of Medicine, Kyoto Background: We established a completely novel method of auxiliary xenogeneic University, Kyoto, Japan partial liver transplantation and examined whether liver grafts procured from Syrian 2Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan hamsters regenerated in nude rats, which were used as in vivo bioreactors. 3Department of Organ Fabrication, Keio Methods: The hamsters and the rats were all males (n = 10). Partial liver grafts from University School of Medicine, Tokyo, Japan hamsters were transplanted into nude rats in an auxiliary manner. We evaluated liver Correspondence graft injury, rejection, and regeneration during 7 days after auxiliary xenogeneic par- Shintaro Yagi, 54 Kawahara‐cho, Shogoin, Sakyo‐ku, Kyoto, 606‐8507, Japan. tial liver transplantation. Email: [email protected]‐u.ac.jp Results: All rats survived until sacrifice on post‐operative day (POD) 1, 3, and 7. HE‐ Funding information staining showed normal at POD1, mild periportal edema, and slight bile duct and Japan Society for the Promotion of Science, venous endothelial inflammation at POD3, and moderate acute cellular rejection at Grant/Award Number: 17H06814 POD7 without parenchymal necrosis.
    [Show full text]
  • Structure of the Trypanosome Cyanide-Insensitive Alternative Oxidase
    Structure of the trypanosome cyanide-insensitive alternative oxidase Tomoo Shibaa,1,2, Yasutoshi Kidoa,1,3, Kimitoshi Sakamotoa,4, Daniel Ken Inaokaa, Chiaki Tsugea, Ryoko Tatsumia, Gen Takahashib, Emmanuel Oluwadare Baloguna,b,c, Takeshi Narad, Takashi Aokid, Teruki Honmae, Akiko Tanakae, Masayuki Inouef, Shigeru Matsuokaf, Hiroyuki Saimotog, Anthony L. Mooreh, Shigeharu Haradab,5, and Kiyoshi Kitaa,5 aDepartment of Biomedical Chemistry, Graduate School of Medicine, and fGraduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; bDepartment of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan; cDepartment of Biochemistry, Ahmadu Bello University, Zaria 2222, Nigeria; dDepartment of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Tokyo 113-8421, Japan; eSystems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045, Japan; gDepartment of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan; and hBiochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom Edited† by John E. Walker, Medical Research Council Mitochondrial Biology Unit, Cambridge, United Kingdom, and approved February 11, 2013 (received for review October 23, 2012) In addition to haem copper oxidases, all higher plants, some algae, mammalian host in the bloodstream, both the cytochrome re- yeasts, molds, metazoans, and pathogenic microorganisms such as spiratory pathway and oxidative phosphorylation disappear and Trypanosoma brucei contain an additional terminal oxidase, the are replaced by the trypanosomal alternative oxidase (TAO), cyanide-insensitive alternative oxidase (AOX). AOX is a diiron car- which functions as the sole terminal oxidase to reoxidize NADH boxylate protein that catalyzes the four-electron reduction of accumulated during glycolysis (5).
    [Show full text]
  • Cyddc-Mediated Reductant Export in Escherichia Coli Controls the Transcriptional Wiring of Energy Metabolism and Combats Nitrosative Stress Louise V
    Biochem. J. (2016) 473, 693–701 doi:10.1042/BJ20150536 693 CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress Louise V. Holyoake*, Stuart Hunt†, Guido Sanguinetti‡, Gregory M. Cook§, Mark J. Howard*, Michelle L. Rowe*, Robert K. Poole† and Mark Shepherd*1 *School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K. †Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K. ‡School of Informatics, The University of Edinburgh, Informatics Forum, 10 Crichton Street, Edinburgh EH8 9AB, U.K. §Department of Microbiology and Immunology, University of Otago, P.O. Box 56, 720 Cumberland Street, Dunedin 9054, New Zealand The glutathione/cysteine exporter CydDC maintains redox CydDC-mediated reductant export promotes protein misfolding, Downloaded from http://portlandpress.com/biochemj/article-pdf/473/6/693/686013/bj4730693.pdf by guest on 02 October 2021 balance in Escherichia coli.AcydD mutant strain was adaptations to energy metabolism and sensitivity to NO. The used to probe the influence of CydDC upon reduced thiol addition of both glutathione and cysteine to the medium was found export, gene expression, metabolic perturbations, intracellular to complement the loss of bd-type cytochrome synthesis in a cydD pH homoeostasis and tolerance to nitric oxide (NO). Loss strain (a key component of the pleiotropic cydDC phenotype), of CydDC was found to decrease extracytoplasmic thiol providing the first direct evidence that CydDC substrates are levels, whereas overexpression diminished the cytoplasmic able to restore the correct assembly of this respiratory oxidase. thiol content. Transcriptomic analysis revealed a dramatic These data provide an insight into the metabolic flexibility of E.
    [Show full text]
  • Transplantation of Engineered Chimeric Liver with Autologous
    ORIGINAL ARTICLE Transplantation of Engineered Chimeric Liver With Autologous Hepatocytes and Xenobiotic Scaffold Toshiyuki Hata, MD,∗† Shinji Uemoto, MD, PhD,∗ Yasuhiro Fujimoto, MD, PhD,∗ Takashi Murakami, MD, PhD,‡ Chise Tateno, PhD,§ Katsutoshi Yoshizato, PhD,§ and Eiji Kobayashi, MD, PhD|| iver transplantation is currently regarded as the most effective Objective: Generation of human livers in pigs might improve the serious treatment for end-stage liver diseases. Because the worldwide shortage of grafts for human liver transplantation, and enable liver trans- L graft shortage remains unresolved,1,2 engineered organs are antici- plantation without the need for deceased or living donors. We developed a pated as alternative grafts to fill the scarcity and ultimately replace chimeric liver (CL) by repopulation of rat hepatocytes in a mouse and suc- those from deceased and living donors. Although regenerative tech- cessfully transplanted it into a rat recipient with vessel reconstruction. This nology has already developed various kinds of regenerative cells and study was designed to investigate the feasibility of CL for supporting the tissues,3,4 they are still insufficient to cure patients with end-stage recipient after auxiliary liver grafting. liver diseases because of the absence of complicated functions and Methods: Hepatocytes from luciferase transgenic or luciferase/LacZ double- limited volume. Therefore, a regenerative liver graft for use as an “or- transgenic rats were transplanted into 20- to 30-day-old urokinase-type gan” is required. To achieve an appropriate 3-dimensional structure plasminogen activator/severe-combined immunodeficiency (uPA/SCID) mice and differentiation to specific tissues in a single organ, application of (n = 40) to create CLs with rat-origin hepatocytes.
    [Show full text]
  • Development of Fluorescent Substrates for Microsomal Epoxide Hydrolase
    Analytical Biochemistry 414 (2011) 154–162 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies ⇑ Christophe Morisseau a, , Maud Bernay a, Aurélie Escaich a, James R. Sanborn a, Jozsef Lango b, Bruce D. Hammock a a Department of Entomology and Cancer Center, University of California – Davis, Davis, CA 95616, USA b Department of Molecular Biosciences, University of California – Davis, Davis, CA 95616, USA article info abstract Article history: The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous Received 18 January 2011 xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is Received in revised form 23 February 2011 associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several Accepted 25 February 2011 forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and Available online 1 March 2011 synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl Keywords: carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors sim- Microsomal epoxide hydrolase ilarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between Fluorescent substrate a-Cyanocarbonate inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the dis- Xenobiotic metabolism covery of structure–activity relationships among mEH inhibitors.
    [Show full text]