Law Tak Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 178-213). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.v / List of Tables --- p.xiii / List of Figures --- p.xiv / Abbreviations --- p.xvii / Preface --- p.1 / Chapter 1 --- All about OTC / Chapter 1.1 --- The OTC Gene --- p.2 / Chapter 1.1.1 --- Mapping --- p.2 / Chapter 1.1.2 --- Isolation --- p.2 / Chapter 1.1.3 --- Structure --- p.2 / Chapter 1.1.4 --- OTC in other species --- p.4 / Chapter 1.2 --- From Gene to Protein --- p.5 / Chapter 1.2.1 --- Synthesis of pOTC --- p.5 / Chapter 1.2.2 --- Transport of pOTC into the mitochondria matrix --- p.6 / Chapter 1.2.3 --- Processing of pOTC into mature OTC --- p.7 / Chapter 1.2.4 --- Assembly of OTC monomer into trimer --- p.8 / Chapter 1.3 --- The OTC Protein --- p.9 / Chapter 1.3.1 --- Structure --- p.9 / Chapter 1.3.2 --- Distribution and location --- p.10 / Chapter 1.3.3 --- OTC in different species and its role --- p.10 / Chapter 1.4 --- Role of OTC in Urea Cycle --- p.14 / Chapter 1.4.1 --- The urea cycle --- p.14 / Chapter 1.4.2 --- Catalytic mechanism of OTC --- p.15 / Chapter 1.5 --- OTCD --- p.17 / Chapter 1.5.1 --- Urea cycle disorders --- p.17 / Chapter 1.5.2 --- OTCD --- p.17 / Chapter 1.5.3 --- Prevalence of OTCD --- p.17 / Chapter 1.6 --- Symptoms of OTCD --- p.19 / Chapter 1.6.1 --- Symptoms --- p.19 / Chapter 1.6.2 --- OTCD in males --- p.20 / Chapter 1.6.3 --- OTCD in females --- p.21 / Chapter 1.7 --- Diagnosis of OTCD --- p.23 / Chapter 1.7.1 --- Diagnosis --- p.23 / Chapter 1.7.2 --- Differential diagnosis --- p.24 / Chapter 1.7.3 --- Carrier testing by protein loading test or allopurinol test --- p.25 / Chapter 1.7.4 --- Prenatal diagnosis --- p.26 / Chapter 1.7.5 --- Preimplantation genetic diagnosis --- p.26 / Chapter 1.8 --- Treatments of OTCD --- p.27 / Chapter 1.8.1 --- Diet and supplements --- p.27 / Chapter 1.8.2 --- Alternative pathway therapy --- p.28 / Chapter 1.8.2.1 --- Sodium phenylbutyrate / phenylacetate --- p.28 / Chapter 1.8.2.2 --- Sodium benzoate --- p.28 / Chapter 1.8.3 --- Dialysis --- p.29 / Chapter 1.8.4 --- Liver transplantation --- p.30 / Chapter 1.8.5 --- Other medications --- p.31 / Chapter 1.8.6 --- Counseling --- p.32 / Chapter 1.8.7 --- Gene therapy --- p.32 / Chapter Part I --- DNA Mutation Analysis of OTCD Patients / Chapter 2 --- Introduction / Chapter 2.1 --- Mutations - Cause of OTCD --- p.35 / Chapter 2.1.1 --- Type of mutations --- p.35 / Chapter 2.1.2 --- How mutations cause OTCD? --- p.35 / Chapter 2.2 --- Properties of OTC Mutations --- p.36 / Chapter 2.2.1 --- Heterogeneity --- p.36 / Chapter 2.2.2 --- Neonatal vs late onset --- p.36 / Chapter 2.2.3 --- Recurrent mutations --- p.36 / Chapter 2.2.4 --- Gonadal mosaicism --- p.36 / Chapter 2.2.5 --- Sporadic mutations --- p.37 / Chapter 2.2.6 --- Polymorphic presentations --- p.37 / Chapter 2.3 --- Polymorphisms in OTC --- p.38 / Chapter 2.4 --- Diagnosis of OTCD by Molecular Genetic Methods --- p.39 / Chapter 2.4.1 --- Restriction fragment length polymorphisms --- p.39 / Chapter 2.4.2 --- Single-strand conformation polymorphism --- p.40 / Chapter 2.4.3 --- Denaturing gradient gel electrophoresis --- p.40 / Chapter 2.4.4 --- Chemical mismatch cleavage --- p.41 / Chapter 2.4.5 --- Linkage analysis --- p.41 / Chapter 2.4.6 --- DNA sequencing --- p.41 / Chapter 2.5 --- Advantages of Molecular Genetic Diagnosis --- p.43 / Chapter 2.5.1 --- Diagnosis --- p.43 / Chapter 2.5.2 --- Carrier testing --- p.43 / Chapter 2.5.3 --- Prenatal diagnosis --- p.43 / Chapter 3 --- Materials & Methods / Chapter 3.1 --- Genomic DNA Extraction from OTCD Patients by QIAamp® DNA Blood Mini Kit --- p.57 / Chapter 3.1.1 --- Materials --- p.58 / Chapter 3.1.1.1 --- Patients --- p.58 / Chapter 3.1.1.2 --- QIAamp® DNA blood mini kit --- p.58 / Chapter 3.1.2 --- Methods --- p.59 / Chapter 3.1.2.1 --- Genomic DNA extraction --- p.59 / Chapter 3.2 --- OTC Exons Amplification by PCR --- p.60 / Chapter 3.2.1 --- Materials --- p.61 / Chapter 3.2.1.1 --- Chemicals and reagents for agarose gel electrophoresis --- p.61 / Chapter 3.2.1.2 --- Chemicals and reagents for PCR --- p.61 / Chapter 3.2.1.3 --- MicroSpińёØ S-400 HR columns --- p.62 / Chapter 3.2.2 --- Methods --- p.63 / Chapter 3.2.2.1 --- Primer design and synthesis --- p.63 / Chapter 3.2.2.2 --- Polymerase chain reaction --- p.63 / Chapter 3.2.2.3 --- PCR product purification --- p.64 / Chapter 3.3 --- DNA sequencing --- p.65 / Chapter 3.3.1 --- Materials --- p.66 / Chapter 3.3.1.1 --- Chemicals and reagents for sequencing --- p.66 / Chapter 3.3.2 --- Methods --- p.67 / Chapter 3.3.2.1 --- Sequencing reaction --- p.67 / Chapter 3.3.2.2 --- Sequencing product purification --- p.67 / Chapter 3.3.2.3 --- Sequencing --- p.67 / Chapter 4 --- Results / Chapter 4.1 --- 7Mutations and 1 Questionable Polymorphism were Identified in OTCD Patients --- p.68 / Chapter 4.11 --- Patient 1 carried an Arg26Gln mutation --- p.70 / Chapter 4.12 --- Patient 2 carried a Leu 111 Pro substitution --- p.72 / Chapter 4.13 --- Patient 3 carried a Glul22Gly mutation --- p.73 / Chapter 4.14 --- Patient 4 carried an Argl 29His mutation --- p.75 / Chapter 4.15 --- Patient 5 carried a Lys 144Term mutation --- p.77 / Chapter 4.16 --- Patient 6 carried a Thrl78Met mutation --- p.78 / Chapter 4.17 --- Patient 7 carried an Asnl98Ile mutation --- p.79 / Chapter 4.18 --- Patient 8 carried an IVS 5 + 1 G→T mutation --- p.80 / Chapter 5 --- Discussion / Chapter 5.1 --- Heterogeneity of OTC mutations --- p.82 / Chapter 5.2 --- Two Novel Mutations were Identified: Asnl98Ile and IVS 5 + 1 G →T --- p.83 / Chapter 5.2.1 --- Asnl98Ile --- p.83 / Chapter 5.2.2 --- IVS5+1G→T --- p.83 / Chapter 5.3 --- "Five Known Mutations were Identified, Four of which were Identified in Chinese for the First Time" --- p.84 / Chapter 5.3.1 --- Arg26Gln --- p.84 / Chapter 5.3.2 --- Glul22Gly --- p.84 / Chapter 5.3.3 --- Argl29His --- p.84 / Chapter 5.3.4 --- Lysl44Term --- p.87 / Chapter 5.3.5 --- Thrl78Met --- p.87 / Chapter 5.4 --- A Questionable Polymorphism was Identified: Leu111 Pro --- p.87 / Chapter 5.5 --- Role of DNA Sequencing as a Direct Diagnosis of OTCD --- p.89 / Chapter 5.6 --- A Questionable Polymorphism: Leu101Phe with Allele Frequency --- p.90 / Chapter Part II --- Protein Expression Study of OTC Mutants / Chapter 6 --- Introduction / Chapter 6.1 --- Site-Directed Mutagenesis --- p.92 / Chapter 6.2 --- Protein Expression Systems --- p.95 / Chapter 6.2.1 --- Bacteria --- p.95 / Chapter 6.2.2 --- Yeast --- p.95 / Chapter 6.2.3 --- Baculovirus --- p.96 / Chapter 6.2.4 --- Mammalian cells --- p.96 / Chapter 6.2.5 --- Cell free expression --- p.97 / Chapter 6.3 --- OTC Enzyme Assay --- p.99 / Chapter 7 --- Materials & Methods / Chapter 7.1 --- Obtaining the OTC Clone --- p.101 / Chapter 7.1.1 --- Materials --- p.102 / Chapter 7.1.1.1 --- Chemicals for preparing low salt LB medium / agar with ZeocinTM --- p.102 / Chapter 7.1.1.2 --- AutoSe´qёØ G-50 --- p.103 / Chapter 7.1.1.3 --- GeneStorm® expression-ready clone --- p.103 / Chapter 7.1.1.4 --- QIAprep® miniprep kit --- p.103 / Chapter 7.1.1.5 --- TempliPhíёØ 100 amplification kit --- p.104 / Chapter 7.1.2 --- Methods / Chapter 7.1.2.1 --- Small-scale preparation of pcDNA3.1/OTC by QIAprep® miniprep kit --- p.105 / Chapter 7.1.2.2 --- Amplification of pcDNA3.1-OTC by TempliPhíёØ --- p.107 / Chapter 7.1.2.3 --- Primer design and synthesis for sequencing --- p.107 / Chapter 7.1.2.4 --- DNA sequencing --- p.108 / Chapter 7.2 --- Entering into the Gateway System --- p.109 / Chapter 7.2.1 --- Materials --- p.110 / Chapter 7.2.1.1 --- Chemicals and reagents for PCR --- p.110 / Chapter 7.2.1.2 --- Chemicals and reagents for preparing LB medium/agar with kanamycin --- p.110 / Chapter 7.2.1.3 --- pENTR Directional TOPO® cloning kit --- p.110 / Chapter 7.2.2 --- Methods --- p.112 / Chapter 7.2.2.1 --- Primer design and synthesis --- p.112 / Chapter 7.2.2.2 --- Polymerase chain reaction --- p.113 / Chapter 7.2.2.3 --- TOPO® cloning reaction --- p.114 / Chapter 7.2.2.4 --- Transformation --- p.114 / Chapter 7.2.2.5 --- Spreading plates --- p.115 / Chapter 7.3 --- Investigation of Subcellular Localization of OTC --- p.116 / Chapter 7.3.1 --- Materials --- p.117 / Chapter 7.3.1.1 --- Chemicals and reagents for cell culture --- p.117 / Chapter 7.3.1.2 --- 4% paraformaldehyde in PBS --- p.118 / Chapter 7.3.1.3 --- Ampicillin --- p.118 / Chapter 7.3.1.4 --- Cells --- p.119 / Chapter 7.3.1.5 --- pcDNA-DEST47 --- p.119 / Chapter 7.3.1.6 --- QuikChange® II XL site-directed mutagenesis kit --- p.119 / Chapter 7.3.1.7 --- LipofectaminéёØ2000 --- p.120 / Chapter 7.3.1.8 --- LR Clonase reaction mix --- p.120 / Chapter 7.3.1.9 --- MitoTracker® Red CMXRos --- p.120 / Chapter 7.3.1.10 --- Nikon Optiphot-2 component microscope --- p.120 / Chapter 7.3.2 --- Methods --- p.121 / Chapter 7.3.2.1 --- Swapping OTC gene from pENTR/D-TOPO to pcDNA-DEST4´7ёØ by LR ClonaséёØ reaction --- p.121 / Chapter 7.3.2.2 --- Site-directed mutagenesis of OTC by QuikChange® II XL site-directed mutagenesis kit --- p.123 / Chapter 7.3.2.2.1 --- Primer design and synthesis --- p.123 / Chapter 7.3.2.2.2 --- Mutant strand synthesis --- p.123 / Chapter 7.3.2.2.3 --- DpnI digestion of parental strand --- p.124 / Chapter 7.3.2.2.4 --- Cloning --- p.124 / Chapter 7.3.2.3 --- Cell culture --- p.125 / Chapter 7.3.2.4 --- Transfection of OTC into Hep3B and HepG2 by LipofectaminéёØ2000 --- p.126 / Chapter 7.3.2.5 --- Staining of mitochondria by MitoTracker® --- p.127 / Chapter 7.3.2.6 --- Fixation of cells --- p.127 / Chapter 7.3.2.7 --- Fluorescence microscopy --- p.128 / Chapter 7.4 --- Investigation of Enzyme Activity of OTC Mutants Expressed in a Cell-free System --- p.129 / Chapter 7.4.1 --- Materials --- p.130 / Chapter 7.4.1.1 --- Chemical and reagents for His-tag protein stain --- p.130 / Chapter 7.4.1.2 --- Chemicals and reagents for OTC enzyme assay --- p.130 / Chapter 7.4.1.3 --- Chemicals and reagents for NuPAGE® Novex pre-cast gel system --- p.131 / Chapter 7.4.1.4 --- Chemicals and reagents for total protein stain --- p.132 / Chapter 7.4.1.5 --- Chemicals and reagents for Tris-Glycine SDS-PAGE --- p.132 / Chapter 7.4.1.6 --- ExpressWayT M plus expression system --- p.134 / Chapter 7.4.1.7 --- pEXPl-DEST vector --- p.134 / Chapter 7.4.1.8 --- β-Gal assay kit --- p.135 / Chapter 7.4.1.9 --- Rapid translation system RTS GroE supplement --- p.135 / Chapter 7.4.2 --- Methods --- p.136 / Chapter 7.4.2.1 --- Swapping OTC gene from pENTR/D-TOPO to pEXPl-DEST by LR Clonase reaction --- p.136 / Chapter 7.4.2.2 --- Site-directed mutagenesis of pEXP1-DEST/OTC by QuikChange® II XL site-directed mutagenesis kit --- p.136 / Chapter 7.4.2.3 --- Cell-free expression by ExpressWayT M plus expression system --- p.137 / Chapter 7.4.2.4 --- Preparation of proteins for SDS PAGE --- p.138 / Chapter 7.4.2.5 --- SDS-PAGE --- p.138 / Chapter 7.4.2.6 --- Staining of His-tagged fusion protein by In VisiońёØ His-tag In-gel stain --- p.139 / Chapter 7.4.2.7 --- OTC enzyme assay --- p.140 / Chapter 7.4.2.7.1 --- Validation of OTC enzyme assay by normal subjects' sera --- p.140 / Chapter 7.4.2.7.2 --- Determination of linear range by OTC and citrulline --- p.140 / Chapter 7.4.2.7.3 --- Enzyme assay of cell-free expressed WT OTC --- p.140 / Chapter 7.4.2.8 --- β -Gal assay --- p.141 / Chapter 8 --- Results / Chapter 8.1 --- The OTC Gene in GeneStorm® Expression-Ready Clone Showed 3 Mismatches with the Published OTC cDNA Sequence (GenBank Accession Number NM_00531) --- p.143 / Chapter 8.2 --- OTC and its Mutants Showed a Mitochondrial Distribution in Hep3B and HepG2 --- p.145 / Chapter 8.2.1 --- pcDNA-DEST47/OTC with desired mutations generated --- p.147 / Chapter 8.2.2 --- OTC and its mutants showed a mitochondrial distribution in Hep3B --- p.149 / Chapter 8.2.3 --- OTC and its mutants showed a mitochondrial distribution in HepG2 --- p.151 / Chapter 8.3 --- Cell-free Expression is not a Feasible Method for Expressing Active OTC --- p.153 / Chapter 8.3.1 --- pEXPl/OTC2 with desired mutations generated --- p.156 / Chapter 8.3.2 --- OTC and its mutants were expressed by the cell-free system as shown in SDS-PAGE analysis --- p.158 / Chapter 8.3.3 --- OTC and its mutants expression were confirmed by His-Tag In-gel stain --- p.160 / Chapter 8.3.4 --- Setup of OTC assay was validated --- p.161 / Chapter 8.3.4.1 --- Validation of OTC enzyme assay with normal subjects' sera --- p.161 / Chapter 8.3.4.2 --- Establishment of the linear relationship of enzymatic reaction in Streptococcus faecalis OTC enzyme assay --- p.163 / Chapter 8.3.4.3 --- Establishment of the linear relationship of colorimetric reaction in OTC enzyme assay --- p.165 / Chapter 8.3.4.4 --- OTC synthesized by cell-free expression system was not active --- p.167 / Chapter 9 --- Discussion / Chapter 9.1 --- "Mutations in OTC, Including Arg26Gln, have no Effect on the Subcellular Localization of OTC in Hep3B and HepG2" --- p.169 / Chapter 9.1.1 --- Arg26Gln --- p.170 / Chapter 9.1.2 --- "Leu 101 Phe, Leu111 Pro, Thrl78Met, and Asnl98Ile" --- p.172 / Chapter 9.2 --- Arg129His Mutant may be unstable --- p.173 / Chapter 9.3 --- Cell-free Expression may not be a Feasible Method for Expression of Active OTC --- p.174 / Bibliography --- p.178 / Appendices / Chapter A.1 --- DNA Sequence of OTC --- p.214 / Chapter A.2 --- Amino Acid Sequence of OTC --- p.215 / Chapter A.3 --- Splicing Sites in OTC --- p.216 / Chapter A.4 --- Multiple Alignments of OTC Protein from 45 Species --- p.217 / Chapter A.5 --- Summary of Patients' Information --- p.218 / Chapter A.6 --- Primers Used in PCR and Sequencing of Patients' Genomic DNA and the Sequence Amplified --- p.223 / Chapter A.7 --- Primers Used in GeneStorm© Expression-Ready Clone and the Sequence Amplified --- p.227 / Chapter A.8 --- Primers Used in pENTR Directional TOPO® Cloning Kit --- p.228 / Chapter A.9 --- Primers Used in Mutagenesis and the Codon Changed --- p.229 / Chapter A.10 --- Vector Information of pcDNA-DEST47 --- p.231 / Chapter A.11 --- Vector Information of pcDNA/GW-47/CAT --- p.232 / Chapter A.12 --- Vector Information of pcDNA3.1/GS --- p.233 / Chapter A.13 --- Vector Information of pEXPl-DEST --- p.234 / Chapter A.14 --- Vector Information of pEXPl-GW/lacZ --- p.235 / Chapter A.15 --- Vector Information of pENTR/D-TOPO --- p.236 / Chapter A.16 --- Vector Information of pIVEX Control Vector GFP --- p.237 / Chapter A.17 --- Genotype of Bacteria Cells --- p.238 / Chapter A.18 --- Details of Markers --- p.239

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 1

J'ai mis au point une méthodologie pour annoter les superfamilles d'enzymes, en décrire l'histoire et les replacer dans l'évolution de leurs voies métaboliques. J'en ai étudié trois : (1) les amidohydrolases cycliques, dont les DHOases (dihydroorotases, biosynthèse des pyrimidines), pour lesquelles j'ai proposé une nouvelle classification. L'arbre phylogénétique inclut les dihydropyrimidinases (DHPases) et allantoïnases (ALNases) qui ont des réactions similaires dans d'autres voies (dégradation des pyrimidines et des purines respectivement). (2) L'étude de la superfamille des DHODases (qui suivent les DHOases) montre une phylogénie semblable aux DHOases, avec également des enzymes d'autres voies, dont les DHPDases (qui suivent les DHPases). De cette observation est né le concept de module réactionnel, qui correspond à la conservation de l’enchaînement de réactions semblables dans différentes voies métaboliques. Cela a été utilisé lors de (3) l'étude des carbamoyltransférases (TCases) qui incluent les ATCases (précédant les DHOases). J'ai d'abord montré l'existence d'une nouvelle TCase potentiellement impliquée dans la dégradation des purines et lui ai proposé un nouveau rôle en utilisant le concept de module réactionnel (enchaînement avec l'ALNase). Dans ces trois grandes familles j'ai aussi mis en évidence trois groupes de paralogues non identifiés qui se retrouvent pourtant dans un même contexte génétique appelé « Yge » et qui formeraient donc un module réactionnel constitutif d'une nouvelle voie hypothétique. Appliqué à diverses voies, le concept de modules réactionnels refléterait donc les voies métaboliques ancestrales dont ils seraient les éléments de base. / I designed a methodology to annotate enzyme superfamilies, explain their history and describe them in the context of metabolic pathways evolution. Three superfamilies were studied: (1) cyclic amidohydrolases, including DHOases (dihydroorotases, third step of the pyrimidines biosynthesis), for which I proposed a new classification. The phylogenetic tree also includes dihydropyrimidinases (DHPases) and allantoinases (ALNases) which catalyze similar reactions in other pathways (pyrimidine and purine degradation, respectively). (2) The DHODases superfamily (after DHOases) show a similar phylogeny as DHOases, including enzymes from other pathways, DHPDases in particular (after DHPases). This led to the concept of reaction module, i.e. a conserved series of similar reactions in different metabolic pathways. This was used to study (3) the carbamoyltransferases (TCases) which include ATCases (before DHOases). I first isolated a new kind of TCase, potentially involved in the purine degradation, and I proposed a new role for it in the light of reaction modules (linked with ALNase). In those three superfamilies I also found three groups of unidentified paralogs that were remarkably part of the same genetic context called “Yge” which would be a reaction module part of an unidentified pathway. The concept of reactions modules may then reflect the ancestral metabolic pathways for which they would be basic elements.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple sclerosis (MS) is a disabling disease that afflicts an estimated two million people worldwide. The disease is characterized by degradation of the myelin sheath that insulates neurons of the central nervous system manifesting as a heterogeneous collection of symptoms. Two enzymes, protein arginine deaminases type 2 and 4 (PAD2 and PAD4) have been implicated to play an etiologic role in demyelination and neurodegeneration by catalyzing a post-translational modification of arginine peptide residues to citrulline. The pathogenesis of MS is poorly understood, though vitamin D deficiency is a well-associated risk factor for developing the disorder. Using the experimental autoimmune encephalomyelitis (EAE) model of MS we demonstrate vitamin D treatment to attenuate over-expression of PAD 2 and 4 in the brain and spine during EAE. In addition, we identify two molecules produced by peripheral immune cells, IFNɣ and IL-6, as candidate signaling molecules that induce PAD expression in the brain. We demonstrate vitamin D treatment to inhibit IFNɣ mediated up regulation of PAD2 and PAD4 both directly within the brain and by modulating PAD-inducing cytokine production by infiltrating immune cells. These results provide neuroprotective rational for the supplementation of vitamin D in MS patients. More importantly, these results imply an epigenetic link between vitamin D deficiency and the pathogenesis of MS that merits further investigation.