DOCSLIB.ORG

Proteome Characterization of Caenorhabditis Elegans During

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

PROTEOME CHARACTERIZATION OF CAENORHABDITIS ELEGANS DURING DEVELOPMENTAL STAGES by ASIFA KHATOON ZAIDI Submitted in partial fulfillment of the requirements for the degree of Master of Science Systems Biology And Bioinformatics CASE WESTERN RESERVE UNIVERSITY May 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Asifa Khatoon Zaidi Candidate for the degree of Master of Science Committee Chair Dr. Masaru Miyagi Committee Member Dr. David Lodowski Committee Member Dr. Gurkan Bebek Date of Defense March 25, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein 2 Table of Contents Table of Contents 3 List of Tables 6 List of Figures 10 Acknowledgments 12 Abstract 13 A: CHAPTER 1: Proteome characterization of Caenorhabditis elegans at different developmental stages 14 1. Summary 14 2. Introduction 15 3. Methods and material 18 3.1. Materials 18 3.2. Methods 18 3.2.1. C. elegans Strain, Maintenance, and Age Synchronization 18 3.2.1.1. Preparation of Nematode Growth Media (NGM) plates 19 3.2.1.2. Equipment and reagents 19 3.2.1.3. Methods 19 12 13 3.3. Labeling Bacteria with Light-( C6-Lys) and Heavy-( C6-Lys) Lysine 19 3.4. Preparation of labeled C. elegans at different larval stages 20 3.5. Separation of Live and Dead Worms 21 3.6. Sample preparation for proteomic analysis 21 3.7. LC-MS/MS analysis 23 3.8. Identification and Quantification of Peptides and Proteins 25 3 3.9. Data analysis 26 4. Results 27 4.1. Overview of the data set 27 4.2. Clustering analysis 29 4.2.1. Principal component analysis (PCA) 29 4.2.2. Unsupervised clustering analysis 31 4.3. Overall distribution appearance rate of newly synthesized proteins 35 4.4. Gene ontology and pathway association of the proteins for which PAR was rapid at different development stages 37 4.4.1. Gene ontology: molecular function 37 4.4.2. Gene ontology: biological function 44 4.4.3. Pathways (KEGG) association 51 5. Discussion 59 B. CHAPTER 2: Effect of environmental stress on proteome of Caenorhabditis elegans during development 62 1. Summary 62 2. Introduction 63 3. Materials and methods 66 3.1. Preparation of unlabeled C. elegans L3 larvae 66 3.2. Preparation of labeled C. elegans dauer larvae 66 13 3.3. Preparation of C6-Lys-labeled reference L3 and dauer larvae 68 3.4. Data analysis 68 4. Results 69 4 4.1. Overall expression of total proteins in dauer and L3 larvae 69 4.2. Clustering analysis 71 4.2.1. Principal component analysis (PCA) 71 4.2.2. Unsupervised clustering analysis 74 4.3. Differentially expressed proteins between L3 and dauer larva 76 4.4. Gene ontology and pathway association 79 4.5.1. Gene Ontology and Pathway Association of up regulated proteins in dauer larvae 79 4.5.2. Gene Ontology and Pathway Association of down regulated proteins in dauer larvae 94 4.5.3. Gene Ontology and Pathway Association of unique proteins present in dauer larvae 118 4.5.4. Gene Ontology and Pathway Association of unique proteins in present in L3 larvae 128 5. Discussion 152 C. Appendix 155 D. Bibliography 181 5 List of Tables CHAPTER 1 Table-1.1: Gene ontology (GO)-molecular function (MF) for proteins with rapid PAR at different developmental stages. 40 Table-1.2: The statistical overrepresentation test (PANTHER GO-Molecular Function) of proteins with rapid PAR at different development stages. 43 Table-1.3: Gene ontology (GO): Biological Process (BP) for proteins with rapid PAR at different developmental stages. 46 Table-1.4: The statistical overrepresentation test (PANTHER GO-Biological Process) of proteins with rapid PAR at different development stages. 50 Table-1.5: The pathway (KEGG) association for proteins with rapid PAR at L1→L2 stages. 55 Table-1.6: The pathway (KEGG) association for the proteins with rapid PAR at L2→L3 stages. 56 Table-1.7: The pathway (KEGG) association for the proteins with rapid PAR at L4→YA stages. 57 Table-1.8: The pathway (KEGG) association for the proteins with rapid PAR at L4→YA stages. 58 CHAPTER 2 Table-2.1: Proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm 83 6 Table -2.2: GO-MF terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm 84 Table-2.3: GO-BP terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 85 Table-2.4: Statistical overrepresentation test (PANTHER) for GO-MF and GO-BP terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 86 Table-2.5: KEGG pathways for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 88 Table-2.6: REACTOME pathways for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 89 Table-2.7: Proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm 98 Table-2.8: GO-MF terms for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm 102 Table-2.9: GO-BP terms for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. 106 Table-2.10: KEGG pathway for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. 114 Table-2.11: REACTOME overrepresentation pathway for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. 117 Table-2.12: Unique proteins present in dauer. 120 Table-2.13: GO-MF terms for the unique proteins in dauer. 121 7 Table-2.14: GO-BP terms for the unique proteins in dauer. 122 Table-2.15: KEGG pathway for the unique proteins in dauer. 126 Table-2.16: REACTOME overrepresentation pathway for the unique proteins in dauer. 127 Table-2.17: Unique proteins present in L3. 130 Table-2.18: GO-MF terms for the unique proteins in L3. 135 Table-2.19: GO-BP terms for the unique proteins in L3. 140 Table-2.20: KEGG pathway for the unique proteins in L3. 145 Table-2.21: REACTOME overrepresentation pathway for the unique proteins in L3. 151 APPENDIX Supplementary Table-1.1: Proteins for which PAR is rapid at L1→L2 stage 155 Supplementary Table-1.2: Proteins for which PAR is rapid at L2→L3 stage 156 Supplementary Table-1.3: Proteins for which PAR is rapid at L3→L4 stage 158 Supplementary Table-1.4: Proteins for which PAR is rapid at L4 →YA stage 161 Supplementary Table-1.5: Proteins for which PAR is slow at L1→L2 stage 164 Supplementary Table-1.6: Proteins for which PAR is slow at L2→L3 stage 166 Supplementary Table-1.7: Proteins for which PAR is slow at L3→L4 stage 168 Supplementary Table-1.8: Proteins for which PAR is slow at L4 →YA stage 169 Supplementary Table-1.9: Gene ontology (GO): molecular function (MF) for proteins with slow PAR at different developmental stages. 170 Supplementary Table-1.10: The Statistical overrepresentation test (PANTHER GO- Molecular Function) of proteins with slow PAR at different development stages. 172 8 Supplementary Table-1.11: Gene ontology (GO): Biological Process (BP) for proteins with slow PAR at different developmental stages. 173 Supplementary Table-1.12: Statistical overrepresentation test (PANTHER GO-Biological Process) of proteins with slow PAR at different development stages. 176 Supplementary Table-1.13: KEGG pathway for proteins with slow PAR at L1→L2 stages. 177 Supplementary Table-1.14: KEGG pathway for proteins with slow PAR at L2→L3 stages. 178 Supplementary Table-1.15: KEGG pathway for proteins with slow PAR at L4→YA stages. 179 Supplementary Table-1.16: KEGG pathway for proteins with slow PAR at L4→YA stages. 180 9 List of Figures CHAPTER 1 Figure 1.1: Experimental Workflow 22 Figure 1.2: Proteins identified during different developmental stages 28 Figure 1.3: Principle component analysis (PCA) of the proteins identified during different development stage. 30 Figure 1.4: Scree plot depicting variance of the all variance of the principle component axis’s present in the different development stage. 33 Figure 1.5: Heat map of the proteins identified during different development stage. 34 Figure 1.6: Overall distribution of the PAR measured for identified proteins during different larval development stages 36 Figure 1.7: Gene Ontology (GO)-Molecular function (MF) terms for proteins with rapid PAR. 39 Figure 1.8: Gene Ontology-Biological Process terms for proteins with rapid PAR. 45 Figure 1.9: Pathway (KEGG) association for proteins with rapid PAR mapped to metabolic process. 54 CHAPTER 2 Figure 2.1: Overview of workflow 67 Figure 2.2: Overall distribution of newly synthesized proteins at L3 and dauer larval stages 70 Figure 2.3: Principal component analysis (PCA) 72 10 Figure 2.4: Scree plot depicting variance of the all principle component axis’s present in the L3 and dauer larvae samples in triplicates. 73 Figure 2.5: Heat map 75 Figure 2.6: Volcano plot 77 Figure 2.7: Fold change of proteins in dauer larvae to L3 Larvae 78 Figure 2.8: Gene Ontology-Molecular Function terms for 1.5-fold up regulated dauer proteins.
Recommended publications
  • Supplementary Data

    Supplementary Data

    Supplementary Data for Quantitative Changes in the Mitochondrial Proteome from Subjects with Mild Cognitive Impairment, Early Stage and Late Stage Alzheimer’s disease Table 1 - 112 unique, non-redundant proteins identified and quantified in at least two of the three analytical replicates for all three disease stages. Table 2 - MCI mitochondrial samples, Protein Summary Table 3 - MCI mitochondrial samples, Experiment 1 Table 4 - MCI mitochondrial samples, Experiment 2 Table 5 - MCI mitochondrial samples, Experiment 3 Table 6 - EAD Mitochondrial Study, Protein Summary Table 7 - EAD Mitochondrial Study, Experiment 1 Table 8 - EAD Mitochondrial Study, Experiment 2 Table 9 - EAD Mitochondrial Study, Experiment 3 Table 10 - LAD Mitochondrial Study, Protein Summary Table 11 - LAD Mitochondrial Study, Experiment 1 Table 12 - LAD Mitochondrial Study, Experiment 2 Table 13 - LAD Mitochondrial Study, Experiment 3 Supplemental Table 1. 112 unique, non-redundant proteins identified and quantified in at least two of the three analytical replicates for all three disease stages. Description Data MCI EAD LAD AATM_HUMAN (P00505) Aspartate aminotransferase, mitochondrial precursor (EC Mean 1.43 1.70 1.31 2.6.1.1) (Transaminase A) (Glutamate oxaloacetate transaminase 2) [MASS=47475] SEM 0.07 0.09 0.09 Count 3.00 3.00 3.00 ACON_HUMAN (Q99798) Aconitate hydratase, mitochondrial precursor (EC 4.2.1.3) Mean 1.24 1.61 1.19 (Citrate hydro-lyase) (Aconitase) [MASS=85425] SEM 0.05 0.17 0.18 Count 3.00 2.00 3.00 ACPM_HUMAN (O14561) Acyl carrier protein, mitochondrial
  • IMPDH2: a New Gene Associated with Dominant Juvenile-Onset Dystonia-Tremor Disorder

    IMPDH2: a New Gene Associated with Dominant Juvenile-Onset Dystonia-Tremor Disorder

    www.nature.com/ejhg BRIEF COMMUNICATION OPEN IMPDH2: a new gene associated with dominant juvenile-onset dystonia-tremor disorder 1,8 1,8 2 3 1,4 2 5 Anna Kuukasjärvi , Juan✉ C. Landoni , Jyrki Kaukonen , Mika Juhakoski , Mari Auranen , Tommi Torkkeli , Vidya Velagapudi and Anu Suomalainen 1,6,7 © The Author(s) 2021 The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenasegene(IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia. European Journal of Human Genetics; https://doi.org/10.1038/s41431-021-00939-1 INTRODUCTION The disease-onset was between 9 and 20 years of age. Table 1 Dystonias are rare movement disorders characterised by sustained or summarises the clinical presentations. intermittent muscle contractions causing abnormal, often repetitive, movements and/or postures. Dystonia can manifest as an isolated Case report symptom or combined with e.g. parkinsonism or myoclonus [1]. While Patient II-6 is a 46-year-old woman.
  • Table 2. Significant

    Table 2. Significant

    Table 2. Significant (Q < 0.05 and |d | > 0.5) transcripts from the meta-analysis Gene Chr Mb Gene Name Affy ProbeSet cDNA_IDs d HAP/LAP d HAP/LAP d d IS Average d Ztest P values Q-value Symbol ID (study #5) 1 2 STS B2m 2 122 beta-2 microglobulin 1452428_a_at AI848245 1.75334941 4 3.2 4 3.2316485 1.07398E-09 5.69E-08 Man2b1 8 84.4 mannosidase 2, alpha B1 1416340_a_at H4049B01 3.75722111 3.87309653 2.1 1.6 2.84852656 5.32443E-07 1.58E-05 1110032A03Rik 9 50.9 RIKEN cDNA 1110032A03 gene 1417211_a_at H4035E05 4 1.66015788 4 1.7 2.82772795 2.94266E-05 0.000527 NA 9 48.5 --- 1456111_at 3.43701477 1.85785922 4 2 2.8237185 9.97969E-08 3.48E-06 Scn4b 9 45.3 Sodium channel, type IV, beta 1434008_at AI844796 3.79536664 1.63774235 3.3 2.3 2.75319499 1.48057E-08 6.21E-07 polypeptide Gadd45gip1 8 84.1 RIKEN cDNA 2310040G17 gene 1417619_at 4 3.38875643 1.4 2 2.69163229 8.84279E-06 0.0001904 BC056474 15 12.1 Mus musculus cDNA clone 1424117_at H3030A06 3.95752801 2.42838452 1.9 2.2 2.62132809 1.3344E-08 5.66E-07 MGC:67360 IMAGE:6823629, complete cds NA 4 153 guanine nucleotide binding protein, 1454696_at -3.46081884 -4 -1.3 -1.6 -2.6026947 8.58458E-05 0.0012617 beta 1 Gnb1 4 153 guanine nucleotide binding protein, 1417432_a_at H3094D02 -3.13334396 -4 -1.6 -1.7 -2.5946297 1.04542E-05 0.0002202 beta 1 Gadd45gip1 8 84.1 RAD23a homolog (S.
  • The Intrinsically Disordered Protein SPE-18 Promotes Localized Assembly of MSP in Caenorhabditis Elegans Spermatocytes Kari L

    The Intrinsically Disordered Protein SPE-18 Promotes Localized Assembly of MSP in Caenorhabditis Elegans Spermatocytes Kari L

    © 2021. Published by The Company of Biologists Ltd | Development (2021) 148, dev195875. doi:10.1242/dev.195875 RESEARCH ARTICLE The intrinsically disordered protein SPE-18 promotes localized assembly of MSP in Caenorhabditis elegans spermatocytes Kari L. Price*,¶, Marc Presler‡,¶, Christopher M. Uyehara§ and Diane C. Shakes ABSTRACT Buracco et al., 2019; Brouhard and Rice, 2018; Bodakuntla et al., Many specialized cells use unconventional strategies of cytoskeletal 2019; de Forges et al., 2012). However, a full understanding of control. Nematode spermatocytes discard their actin and tubulin cytoskeletal control requires consideration of less-studied proteins following meiosis, and instead employ the regulated assembly/ whose properties challenge our standard assumptions. disassembly of the Major Sperm Protein (MSP) to drive sperm One such protein is the nematode Major Sperm Protein (MSP), motility. However, prior to the meiotic divisions, MSP is sequestered assembly/disassembly dynamics of which power the crawling through its assembly into paracrystalline structures called fibrous motility of nematode spermatozoa (Klass and Hirsh, 1981; bodies (FBs). The accessory proteins that direct this sequestration Sepsenwol et al., 1989; Italiano et al., 1996; reviewed by Roberts process have remained mysterious. This study reveals SPE-18 as an and Stewart, 2012; Smith, 2014). Although MSP-based motility intrinsically disordered protein that is essential for MSP assembly appears superficially similar to its actin-based counterpart, the within FBs. In spe-18 mutant spermatocytes, MSP forms disorganized molecular mechanisms are distinct. Much of what we know about cortical fibers, and the cells arrest in meiosis without forming haploid MSP dynamics was gleaned from the parasitic nematode Ascaris, sperm.
  • (12) United States Patent (10) Patent No.: US 8,603,824 B2 Ramseier Et Al

    (12) United States Patent (10) Patent No.: US 8,603,824 B2 Ramseier Et Al

    USOO8603824B2 (12) United States Patent (10) Patent No.: US 8,603,824 B2 Ramseier et al. (45) Date of Patent: Dec. 10, 2013 (54) PROCESS FOR IMPROVED PROTEIN 5,399,684 A 3, 1995 Davie et al. EXPRESSION BY STRAIN ENGINEERING 5,418, 155 A 5/1995 Cormier et al. 5,441,934 A 8/1995 Krapcho et al. (75) Inventors: Thomas M. Ramseier, Poway, CA 5,508,192 A * 4/1996 Georgiou et al. .......... 435/252.3 (US); Hongfan Jin, San Diego, CA 5,527,883 A 6/1996 Thompson et al. (US); Charles H. Squires, Poway, CA 5,558,862 A 9, 1996 Corbinet al. 5,559,015 A 9/1996 Capage et al. (US) 5,571,694 A 11/1996 Makoff et al. (73) Assignee: Pfenex, Inc., San Diego, CA (US) 5,595,898 A 1/1997 Robinson et al. 5,610,044 A 3, 1997 Lam et al. (*) Notice: Subject to any disclaimer, the term of this 5,621,074 A 4/1997 Bjorn et al. patent is extended or adjusted under 35 5,622,846 A 4/1997 Kiener et al. 5,641,671 A 6/1997 Bos et al. U.S.C. 154(b) by 471 days. 5,641,870 A 6/1997 Rinderknecht et al. 5,643,774 A 7/1997 Ligon et al. (21) Appl. No.: 11/189,375 5,662,898 A 9/1997 Ligon et al. (22) Filed: Jul. 26, 2005 5,677,127 A 10/1997 Hogan et al. 5,683,888 A 1 1/1997 Campbell (65) Prior Publication Data 5,686,282 A 11/1997 Lam et al.
  • Peroxisomal Functions in the Lung and Their Role in the Pathogenesis of Lung Diseases

    Peroxisomal Functions in the Lung and Their Role in the Pathogenesis of Lung Diseases

    Aus dem Institut für Anatomie und Zellbiologie der Justus-Liebig-Universität Gießen Leiter: Prof. Dr. Eveline Baumgart-Vogt Peroxisomal functions in the lung and their role in the pathogenesis of lung diseases Habilitationsschrift zur Erlangung der Venia legendi des Fachbereichs Medizin der Justus-Liebig-Universität Gießen vorgelegt von Srikanth Karnati Gießen 2018 Die nachfolgende Arbeit nimmt Bezug auf folgende Originalarbeiten: 1. Karnati S*, Graulich T, Oruqaj G, Pfreimer S, Seimetz M, Stamme C, Mariani TJ, Weissmann N, Mühlfeld C, Baumgart-Vogt E (2016). Postnatal development of the secretory cells of the distal airways, the bronchiolar club cells in the mouse lung: stereological and molecular biological studies. Cell and Tissue Research. Jun;364(3):543- 57. 2. Karnati S, Baumgart-Vogt E (2009) PeroXisomes in airway epithelia and future prospects of these organelles for pulmonary cell biology. Histochem Cell Biol. Apr: 131(4):447-54. 3. Karnati S, Lüers G, Pfreimer S and Baumgart-Vogt E (2013) Manganese SuperoXide dismutase 2 (MnSOD) is localized to mitochondria but not in peroXisomes. Histochemistry and Cell Biology, Aug:140(2):105-17 4. Karnati S, Palaniswamy S, Alam MR, Oruqaj G, Stamme C, Baumgart-Vogt E (2015) C22- bronchial and T7-alveolar epithelial cell lines of the immortomouse are eXcellent murine cell culture model systems to study pulmonary peroXisome biology and metabolism. Histochemistry and Cell Biology Mar;145(3):287-304. 5. Oruqaj G§, Karnati S§, Vijayan V, Kotarkonda LK, Boateng E, Zhang W, Ruppert C, Günther A, Shi W, Baumgart-Vogt E (2015) Compromised peroXisomes in idiopathic pulmonary fibrosis, a vicious cycle inducing a higher fibrotic response via TGF-β signaling.
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA Co-Expressed Gene List in LUAD

    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
  • B Number Gene Name Mrna Intensity Mrna

    B Number Gene Name Mrna Intensity Mrna

    sample) total list predicted B number Gene name assignment mRNA present mRNA intensity Gene description Protein detected - Membrane protein membrane sample detected (total list) Proteins detected - Functional category # of tryptic peptides # of tryptic peptides # of tryptic peptides detected (membrane b0002 thrA 13624 P 39 P 18 P(m) 2 aspartokinase I, homoserine dehydrogenase I Metabolism of small molecules b0003 thrB 6781 P 9 P 3 0 homoserine kinase Metabolism of small molecules b0004 thrC 15039 P 18 P 10 0 threonine synthase Metabolism of small molecules b0008 talB 20561 P 20 P 13 0 transaldolase B Metabolism of small molecules chaperone Hsp70; DNA biosynthesis; autoregulated heat shock b0014 dnaK 13283 P 32 P 23 0 proteins Cell processes b0015 dnaJ 4492 P 13 P 4 P(m) 1 chaperone with DnaK; heat shock protein Cell processes b0029 lytB 1331 P 16 P 2 0 control of stringent response; involved in penicillin tolerance Global functions b0032 carA 9312 P 14 P 8 0 carbamoyl-phosphate synthetase, glutamine (small) subunit Metabolism of small molecules b0033 carB 7656 P 48 P 17 0 carbamoyl-phosphate synthase large subunit Metabolism of small molecules b0048 folA 1588 P 7 P 1 0 dihydrofolate reductase type I; trimethoprim resistance Metabolism of small molecules peptidyl-prolyl cis-trans isomerase (PPIase), involved in maturation of b0053 surA 3825 P 19 P 4 P(m) 1 GenProt outer membrane proteins (1st module) Cell processes b0054 imp 2737 P 42 P 5 P(m) 5 GenProt organic solvent tolerance Cell processes b0071 leuD 4770 P 10 P 9 0 isopropylmalate
  • Placenta-Derived Exosomes Continuously Increase in Maternal

    Placenta-Derived Exosomes Continuously Increase in Maternal

    Sarker et al. Journal of Translational Medicine 2014, 12:204 http://www.translational-medicine.com/content/12/1/204 RESEARCH Open Access Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy Suchismita Sarker1, Katherin Scholz-Romero1, Alejandra Perez2, Sebastian E Illanes1,2,3, Murray D Mitchell1, Gregory E Rice1,2 and Carlos Salomon1,2* Abstract Background: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies. Methods: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP).
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al

    (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al

    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
  • Identification of the Binding Site for Ammonia in GMP Reductase

    Identification of the Binding Site for Ammonia in GMP Reductase

    Identification of the binding site for ammonia in GMP reductase Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biology Lizbeth Hedstrom, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Molecular and Cell Biology by Tianjiong Yao February 2015 Copyright by Tianjiong Yao © 2015 ABSTRACT Identification of the binding site for ammonia in GMP reductase A thesis presented to the Department of Biology Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Tianjiong Yao The overall reaction of guanosine monophosphate reductase (GMPR) converts GMP to IMP by using NADPH as a cofactor and it includes two sub-steps: (1) a deamination step that releases ammonia from GMP and forms the intermediate E-XMP*; (2) a hydride transfer step that converts E-XMP* to IMP along with the oxidation of NADPH. The hydride transfer step is the rate limiting step, yet we failed to observe a burst of ammonia release. Meanwhile ammonia cannot stay in the same place where it is formed otherwise it will block NADPH. This observation suggests that ammonia remains bound to the enzyme during the hydride transfer step and there exists ammonia holding site after its release from the formation site. We identified a possible ammonia holding site by inspection of crystal structure of human GMPR type 2. Three candidate amino acids were selected and probed by site directed mutagenesis. The substitutions of all three residues decreased the reduction of GMP at least 50 fold and the oxidation of IMP at least 40 fold, and reduced the intermediate production at least 2 fold.
  • In TCR-Stimulated Thymocytes Induction of Chromosomal DNA Degradation Caspase-Activated Deoxyribonuclease in the Possible Involv

    In TCR-Stimulated Thymocytes Induction of Chromosomal DNA Degradation Caspase-Activated Deoxyribonuclease in the Possible Involv

    Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes This information is current as of September 28, 2021. Takuya Nagata, Hiroyuki Kishi, Qing Li Liu, Tomoyasu Yoshino, Tadashi Matsuda, Zhe Xiong Jin, Kimie Murayama, Kazuhiro Tsukada and Atsushi Muraguchi J Immunol 2000; 165:4281-4289; ; doi: 10.4049/jimmunol.165.8.4281 Downloaded from http://www.jimmunol.org/content/165/8/4281 References This article cites 42 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/8/4281.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes1 Takuya Nagata,* Hiroyuki Kishi,* Qing Li Liu,* Tomoyasu Yoshino,* Tadashi Matsuda,* Zhe Xiong Jin,* Kimie Murayama,‡ Kazuhiro Tsukada,† and Atsushi Muraguchi2* TCR engagement of immature CD4؉CD8؉ thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus.