DOCSLIB.ORG

Enzymatic Encoding Methods for Efficient Synthesis Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enzymatic Encoding Methods for Efficient Synthesis Of (19) TZZ__T (11) EP 1 957 644 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/10 (2006.01) C12Q 1/68 (2006.01) 01.12.2010 Bulletin 2010/48 C40B 40/06 (2006.01) C40B 50/06 (2006.01) (21) Application number: 06818144.5 (86) International application number: PCT/DK2006/000685 (22) Date of filing: 01.12.2006 (87) International publication number: WO 2007/062664 (07.06.2007 Gazette 2007/23) (54) ENZYMATIC ENCODING METHODS FOR EFFICIENT SYNTHESIS OF LARGE LIBRARIES ENZYMVERMITTELNDE KODIERUNGSMETHODEN FÜR EINE EFFIZIENTE SYNTHESE VON GROSSEN BIBLIOTHEKEN PROCEDES DE CODAGE ENZYMATIQUE DESTINES A LA SYNTHESE EFFICACE DE BIBLIOTHEQUES IMPORTANTES (84) Designated Contracting States: • GOLDBECH, Anne AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DK-2200 Copenhagen N (DK) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • DE LEON, Daen SK TR DK-2300 Copenhagen S (DK) Designated Extension States: • KALDOR, Ditte Kievsmose AL BA HR MK RS DK-2880 Bagsvaerd (DK) • SLØK, Frank Abilgaard (30) Priority: 01.12.2005 DK 200501704 DK-3450 Allerød (DK) 02.12.2005 US 741490 P • HUSEMOEN, Birgitte Nystrup DK-2500 Valby (DK) (43) Date of publication of application: • DOLBERG, Johannes 20.08.2008 Bulletin 2008/34 DK-1674 Copenhagen V (DK) • JENSEN, Kim Birkebæk (73) Proprietor: Nuevolution A/S DK-2610 Rødovre (DK) 2100 Copenhagen 0 (DK) • PETERSEN, Lene DK-2100 Copenhagen Ø (DK) (72) Inventors: • NØRREGAARD-MADSEN, Mads • FRANCH, Thomas DK-3460 Birkerød (DK) DK-3070 Snekkersten (DK) • GODSKESEN, Michael Anders • LUNDORF, Mikkel Dybro DK-2950 Vedbæk (DK) DK-4000 Roskilde (DK) • GLAD, Sanne Schrøder • JAKOBSEN, Søren DK-2750 Ballerup (DK) DK-2000 Frederiksberg (DK) • NEVE, Søren • OLSEN, Eva Kampmann DK-2800 Lyngby (DK) DK-2730 Herlev (DK) • THISTED, Thomas • ANDERSEN, Anne Lee DK-3600 Frederikssund (DK) DK-4100 Ringsted (DK) • KRONBORG, Tine Titilola Akinleminu • HOLTMANN, Anette DK-3500 Værløse (DK) DK-2750 Ballerup (DK) • SAMS, Christian, • HANSEN, Anders Holm DK-3500 Værløse (DK) DK-2400 Copenhagen NV (DK) • FELDING, Jakob • SØRENSEN, Anders Malling DK-2920 Charlottenlund (DK) DK-2860 Søborg (DK) • FRESKGAARD, Per-Ola S-603 79 Norrköping (SE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 957 644 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 957 644 B1 • GOULIAEV, Alex Haahr • HALPIN DAVID R ET AL: "DNA display I. 3670 Veksoe Sjaelland (DK) Sequence-encoded routing of DNA populations." • PEDERSEN, Henrik PLOS BIOLOGY JUL 2004, vol. 2, no. 7, July 2004 2880 Bagsvaerd (DK) (2004-07), page E173, XP002436101 ISSN: 1545-7885 cited in the application (74) Representative: Høiberg A/S • HALPIN DAVID R ET AL: "DNA display II. Genetic St. Kongensgade 59 A manipulation of combinatorial chemistry 1264 Copenhagen K (DK) libraries for small-molecule evolution" PLOS BIOLOGY, XX, XX, vol. 2, no. 7, July 2004 (56) References cited: (2004-07), pages 1022-1030, XP002424416 ISSN: WO-A-00/23458 WO-A-93/20242 1544-9173 cited in the application WO-A-2004/039825 WO-A-2005/058479 WO-A-2006/053571 2 EP 1 957 644 B1 Description Technical Field of the Invention 5 [0001] The present invention relates to bifunctional complexes and methods for synthesising such complexes, as well as to methods for split-and-mix synthesis of different molecules each linked to a single stranded identifier oligonucleotide comprising a plurality of tags identifying the molecule and/or the chemical entities having participated in the synthesis of the molecule. The invention also relates to a method for generating a library of different bifunctional complexes and methods for selecting molecules and/or identifying molecules having a desirable property, such as affinity for a target 10 compound. Background of the Invention [0002] Synthesis methods known as split-and-mix, or split-and-recombine, are known and have been used for the 15 synthesis of different molecules. Split-and-mix methods for synthesising polypeptides and other biochemical polymers have been disclosed e.g. in US 5,723,598 directed to the generation of a library of bifunctional complexes comprising a polypeptide and an identifier oligonucleotide comprising tags in the form of a sequence of nucleotides identifying the amino acids which participated in the formation of the polypeptide. The methods are directed to chemical linkage of tags and do not disclose enzymatic linkage, such as ligation, of the nucleotide tags making up the identifier oligonucleotide. 20 [0003] WO 00/23458 discloses a split-and-mix method in which nucleic acid tags are involved in both molecule syn- thesis and molecule identification. [0004] WO 2004/039825 and WO 2005/058479 disclose split-and-mix methods wherein tags in the form of identifier oligonucleotides are linked enzymatically. The prior art methods do not disclose ligation of a double-stranded oligonu- cleotide substrate comprising a plurality of tags and complementary anti-tags at least partly hybridised to each other, 25 wherein said ligation results in the formation of an identifier oligonucleotide comprising a plurality of consecutive nucle- otides in the form of covalently linked tags, whereas the anti-tags of the double-stranded oligonucleotide substrate are not affected by the action of the ligase, i.e. no anti-tags become covalently linked as a result of the enzymatic ligation of the tag part of the double-stranded oligonucleotide substrate. [0005] Reference is also made to WO2006/053571 disclosing methods for molecule synthesis. 30 Summary of the Invention [0006] There is a need for improved methods for split-and-mix synthesis of libraries of small molecules for e.g. phar- maceutical and other purposes. The small molecules can initially be synthesised as part of a bifunctional complex further 35 comprising an identifier oligonucleotide identifying the reactants which have participated in the synthesis of the small molecule. [0007] The methods of the present invention employs a ligation step wherein the substrate for the ligase is in a double stranded form and wherein the substrate comprises a plurality of tags and at least one or more anti-tags wherein tags and anti-tag(s) is/are at least partly hybridised to each other. The tags are covalently linked as a result of the action of 40 an enzyme comprising a ligase activity on the double stranded substrate, but no anti-tags are covalently linked as a result of said ligase action. [0008] The method facilitates separation of ligated tags and discrete, non-ligated anti-tags because of the size and molecular weight difference between (i) the afore-mentioned single stranded identifier oligonucleotide comprising a plurality of covalently linked tags and (ii) discrete, non-ligated anti-tags. The identifier oligonucleotide comprising the 45 ligated,tags will typically have a length at least about 3 times the length of the individual anti-tags. [0009] In one embodiment, the tags comprise a 5’ phosphate, or a variant ligatable reactive group, whereas anti-tags do not. Therefore, the growing bifunctional complex will comprise a covalently linked "top"- strand to which a number, such as one or more, of shorter and non-ligated anti-tag(s) is/are hybridised/annealed. This enables removal of anti-tag (s), e.g. after all tag additions have been performed (Fig. 1) or after each tag addition has been performed (Fig. 6). 50 [0010] Removal of anti-tags generally increases fidelity and allows extension after purification of the single-stranded oligonucleotide identifier comprising a plurality of ligated tags. Said extension makes possible the use of selection- specific sequences, which improves robustness towards contamination, which is a well-known phenomena when e.g. PCR amplifications are performed. Also, diversification-sequences can be used which diversifies otherwise un-distin- guishable tag combinations. This makes it possible to identify tag combination sequences which may arise during PCR 55 from a single tag combination. It is advantageous to identify such sequences as they may otherwise be interpreted as arising from several molecular entities containing the same tag combination, thus indicating that the specific tag com- bination corresponds to a small molecule with relatively high ability to be retained during subsequent selection procedures. [0011] Also, because of the design of the tags, cross-hybridization between single stranded tags can be reduced or 3 EP 1 957 644 B1 essentially eliminated. This greatly improves the purification process of the bifunctional complexes comprising single stranded identifier oligonucleotides following the synthesis reactions. One problem associated with purification of bi- functional complexes comprising double stranded identifier oligonucleotides is that such identifiers are prone to illegiti- mate hybridization when renaturing conditions are resumed following a purification process under denaturing conditions. 5 [0012] In order to achieve a minimum degree of cross-hybridization between tags in an identifier oligonucleotide, the tags can be designed using a computer algorithm to maximize or optimize the number of mismatches between any oligo pair, e.g. resulting in a minimum of seven mismatches between any two tag tags. This maximizes or optimize fidelity of hybridization and enables use of sorting methods, such as the ones disclosed e.g. in WO 00/23458 (Harbury). Moreover, it increases robustness of decoding by sequencing, e.g. the tag information can be decoded even if sequencing errors 10 occur. [0013] It is also possible to perform a quenching reaction with the reactants and tags.
Load more

Recommended publications
  • Butyrylcholinesterase 'X
    Br. J. Pharmacol. (1993), 108, 914-919 "f" Macmillan Press Ltd, 1993 Degradation of acetylcholine in human airways: role of butyrylcholinesterase 'X. Norel, *M. Angrisani, C. Labat, I. Gorenne, E. Dulmet, *F. Rossi & C. Brink CNRS URA 1159, Centre Chirurgical Marie-Lannelongue, 133 av. de la R6sistance, 92350 Le Plessis-Robinson, France and *Department of Pharmacology and Toxicology, First Faculty of Medicine and Surgery, via Costantinopoli, 16, 80138 Naples, Italy 1 Neostigmine and BW284C51 induced concentration-dependent contractions in human isolated bron- chial preparations whereas tetraisopropylpyrophosphoramide (iso-OMPA) was inactive on airway rest- ing tone. 2 Neostigmine (0.1 pM) or iso-OMPA (100 pM) increased acetylcholine sensitivity in human isolated bronchial preparations but did not alter methacholine or carbachol concentration-effect curves. 3 In the presence of iso-OMPA (10 pM) the bronchial rings were more sensitive to neostigmine. The pD2 values were, control: 6.05 ± 0.15 and treated: 6.91 ± 0.14. 4 Neostigmine or iso-OMPA retarded the degradation of acetylcholine when this substrate was exogenously added to human isolated airways. A marked reduction of acetylcholine degradation was observed in the presence of both inhibitors. Exogenous butyrylcholine degradation was prevented by iso-OMPA (1O pM) but not by neostigmine (O.1 pM). 5 These results suggest the presence of butyrylcholinesterase activity in human bronchial muscle and this enzyme may co-regulate the degradation of acetylcholine in this tissue. Keywords: Human bronchus; butyrylcholinesterase; acetylcholinesterase; acetylcholine; butyrylcholine; tetraisopropylpyrophos- phoramide; neostigmine Introduction The inherent tone observed in human airways is maintained measurement of the degradation of exogenous ACh or butyr- by the local actions of neuronal inputs derived from the ylcholine (BuCh: specific substrate for BChE) were also per- parasympathetic nervous system.
    [Show full text]
  • Viewed Under 23 (B) Or 203 (C) fi M M Male Cko Mice, and Largely Unaffected Magni Cation; Scale Bars, 500 M (B) and 50 M (C)
    BRIEF COMMUNICATION www.jasn.org Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron Camille Ansermet,* Matthias B. Moor,* Gabriel Centeno,* Muriel Auberson,* † † ‡ Dorothy Zhang Hu, Roland Baron, Svetlana Nikolaeva,* Barbara Haenzi,* | Natalya Katanaeva,* Ivan Gautschi,* Vladimir Katanaev,*§ Samuel Rotman, Robert Koesters,¶ †† Laurent Schild,* Sylvain Pradervand,** Olivier Bonny,* and Dmitri Firsov* BRIEF COMMUNICATION *Department of Pharmacology and Toxicology and **Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland; †Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts; ‡Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia; §School of Biomedicine, Far Eastern Federal University, Vladivostok, Russia; |Services of Pathology and ††Nephrology, Department of Medicine, University Hospital of Lausanne, Lausanne, Switzerland; and ¶Université Pierre et Marie Curie, Paris, France ABSTRACT Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is crit- leaves.4 Most recently, Legati et al. have ical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the shown an association between genetic kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical polymorphisms in Xpr1 and primary fa- sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the milial brain calcification disorder.5 How- molecular identity of the protein(s) participating in the basolateral Pi efflux remains ever, the role of XPR1 in the maintenance unknown. Evidence has suggested that xenotropic and polytropic retroviral recep- of Pi homeostasis remains unknown. Here, tor 1 (XPR1) might be involved in this process. Here, we show that conditional in- we addressed this issue in mice deficient for activation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi Xpr1 in the nephron.
    [Show full text]
  • Nietzsche and Psychedelics – Peter Sjöstedt-H –
    Antichrist Psychonaut: Nietzsche and Psychedelics – Peter Sjöstedt-H – ‘… And close your eyes with holy dread, For he on honey-dew hath fed, And drunk the milk of Paradise.’ So ends the famous fragment of Kubla Khan by the Romantic poet, Samuel Taylor Coleridge. He tells us that the poem was an immediate transcription of an opium-induced dream he experienced in 1797. As is known, the Romantic poets and their kin were inspired by the use of psychoactive substances such as opium, the old world’s common pain reliever. Pain elimination is its negative advantage, but its positive attribute lies in the psychedelic (‘mind- revealing’)1 state it can engender – a state described no better than by the original English opium eater himself, Thomas De Quincey: O just and righteous opium! … thou bildest upon the bosom of darkness, out of the fantastic imagery of the brain, cities and temples, beyond the art of Phidias and Praxiteles – beyond the splendours of Babylon and Hekatómpylos; and, “from the anarchy of dreaming sleep,” callest into sunny light the faces of long-buried beauties … thou hast the keys of Paradise, O just, subtle, and mighty opium!2 Two decades following the publication of these words the First Opium War commences (1839) in which China is martially punished for trying to hinder the British trade of opium to the Chinese people. Though opium, derived from the innocent garden poppy Papavar somniferum, may cradle the keys to Paradise it also clutches the keys to Perdition: its addictive thus potentially ruinous nature is commonly known. Today, partly for these reasons, opiates are mostly illegal without license – stringently so in their most potent forms of morphine and heroin.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002
    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
    [Show full text]
  • Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: a Crystallographic, Kinetic and Calorimetric Study
    Article Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study Terrone L. Rosenberry 1, Xavier Brazzolotto 2, Ian R. Macdonald 3, Marielle Wandhammer 2, Marie Trovaslet-Leroy 2,†, Sultan Darvesh 4,5,6 and Florian Nachon 2,* 1 Departments of Neuroscience and Pharmacology, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA; [email protected] 2 Département de Toxicologie et Risques Chimiques, Institut de Recherche Biomédicale des Armées, 91220 Brétigny-sur-Orge, France; [email protected] (X.B.); [email protected] (M.W.); [email protected] (M.T.-L.) 3 Department of Diagnostic Radiology, Dalhousie University, Halifax, NS B3H 4R2, Canada; [email protected] 4 Department of Medical Neuroscience, Dalhousie University, Halifax, NS B3H 4R2, Canada; [email protected] 5 Department of Chemistry, Mount Saint Vincent University, Halifax, NS B3M 2J6, Canada 6 Department of Medicine (Neurology and Geriatric Medicine), Dalhousie University, Halifax, NS B3H 4R2, Canada * Correspondence: [email protected]; Tel.: +33-178-65-1877 † Deceased October 2016. Received: 26 October 2017; Accepted: 27 November 2017; Published: 29 November 2017 Abstract: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE.
    [Show full text]
  • A Systematic Review on the Implications of O-Linked Glycan Branching and Truncating Enzymes on Cancer Progression and Metastasis
    cells Review A Systematic Review on the Implications of O-linked Glycan Branching and Truncating Enzymes on Cancer Progression and Metastasis 1, 1, 1 1,2,3, Rohitesh Gupta y, Frank Leon y, Sanchita Rauth , Surinder K. Batra * and Moorthy P. Ponnusamy 1,2,* 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68105, USA; [email protected] (R.G.); [email protected] (F.L.); [email protected] (S.R.) 2 Fred and Pamela Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 681980-5900, USA 3 Department of Pathology and Microbiology, UNMC, Omaha, NE 68198-5900, USA * Correspondence: [email protected] (S.K.B.); [email protected] (M.P.P.); Tel.: +402-559-5455 (S.K.B.); +402-559-1170 (M.P.P.); Fax: +402-559-6650 (S.K.B. & M.P.P.) Equal contribution. y Received: 21 January 2020; Accepted: 12 February 2020; Published: 14 February 2020 Abstract: Glycosylation is the most commonly occurring post-translational modifications, and is believed to modify over 50% of all proteins. The process of glycan modification is directed by different glycosyltransferases, depending on the cell in which it is expressed. These small carbohydrate molecules consist of multiple glycan families that facilitate cell–cell interactions, protein interactions, and downstream signaling. An alteration of several types of O-glycan core structures have been implicated in multiple cancers, largely due to differential glycosyltransferase expression or activity. Consequently, aberrant O-linked glycosylation has been extensively demonstrated to affect biological function and protein integrity that directly result in cancer growth and progression of several diseases.
    [Show full text]
  • PROTEOMIC ANALYSIS of HUMAN URINARY EXOSOMES. Patricia
    ABSTRACT Title of Document: PROTEOMIC ANALYSIS OF HUMAN URINARY EXOSOMES. Patricia Amalia Gonzales Mancilla, Ph.D., 2009 Directed By: Associate Professor Nam Sun Wang, Department of Chemical and Biomolecular Engineering Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells. These small vesicles (40-100 nm) have been shown to be secreted by most cell types throughout the body. In the kidney, urinary exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane of renal tubular epithelia. Exosomes contain apical membrane and cytosolic proteins and can be isolated using differential centrifugation. The analysis of urinary exosomes provides a non- invasive means of acquiring information about the physiological or pathophysiological state of renal cells. The overall objective of this research was to develop methods and knowledge infrastructure for urinary proteomics. We proposed to conduct a proteomic analysis of human urinary exosomes. The first objective was to profile the proteome of human urinary exosomes using liquid chromatography-tandem spectrometry (LC- MS/MS) and specialized software for identification of peptide sequences from fragmentation spectra. We unambiguously identified 1132 proteins. In addition, the phosphoproteome of human urinary exosomes was profiled using the neutral loss scanning acquisition mode of LC-MS/MS. The phosphoproteomic profiling identified 19 phosphorylation sites corresponding to 14 phosphoproteins. The second objective was to analyze urinary exosomes samples isolated from patients with genetic mutations. Polyclonal antibodies were generated to recognize epitopes on the gene products of these genetic mutations, NKCC2 and MRP4. The potential usefulness of urinary exosome analysis was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I and using the single nucleotide polymorphism in the ABCC4 gene.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • An Efficient Synthesis of Porphyrins with Different Meso Substituents That Avoids Scrambling in Aqueous Media
    An Efficient Synthesis of Porphyrins with Different meso Substituents that Avoids Scrambling in Aqueous Media Agnieszka Nowak-Krol, Rémi Plamont, Gabriel Canard, J.A. Edzang, Daniel T. Gryko, Teodor Silviu Balaban To cite this version: Agnieszka Nowak-Krol, Rémi Plamont, Gabriel Canard, J.A. Edzang, Daniel T. Gryko, et al.. An Efficient Synthesis of Porphyrins with Different meso Substituents that Avoids Scrambling inAque- ous Media. Chemistry - A European Journal, Wiley-VCH Verlag, 2015, 21 (4), pp.1488-1498. 10.1002/chem.201403677. hal-01130057 HAL Id: hal-01130057 https://hal.archives-ouvertes.fr/hal-01130057 Submitted on 7 Feb 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. An Efficient Synthesis of Porphyrins with Different Meso Substituents that Avoids Scrambling in Aqueous Media Agnieszka Nowak-Król,a,† Rémi Plamont,b,† Gabriel Canard,b,c Judicaelle Andeme Edzang,b,c Daniel T. Grykoa,* and Teodor Silviu Balabanb,* To the memory of Alan Roy Katritzky, magister of heterocyclic chemistry [a] Institute of Organic Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland E-mail: [email protected] [b] Aix Marseille Université, Centrale Marseille, CNRS, Institut des Sciences Moléculaires de Marseille (iSm2), UMR 7313, Chirosciences, Avenue Escadrille Normandie Niemen, St.
    [Show full text]
  • Investigation of Plasma Metabolomics and Neurotransmitter Dysfunction In
    RSC Advances View Article Online PAPER View Journal | View Issue Investigation of plasma metabolomics and neurotransmitter dysfunction in the process of Cite this: RSC Adv.,2019,9,18308 Alzheimer's disease rat induced by amyloid beta 25- 35 Mengying Wei,ab Yuanyuan Liu,a Zifeng Pi, b Kexin Yue,a Shizhe Li,c Mingxin Hu,a Zhiqiang Liu,b Fengrui Song b and Zhongying Liu*a Alzheimer's disease (AD) has become one of the major diseases endangering the health of the elderly. Clarifying the features of each AD animal model is valuable for understanding the onset and progression of diseases and developing potential treatments in the pharmaceutical industry. In this study, we aimed to clarify plasma metabolomics and neurotransmitter dysfunction in the process of AD model rat induced by amyloid beta 25-35 (Ab 25-35). Firstly, Morris Water Maze (MWM) test was used to investigate cognitive impairment in AD rat after 2, 4 and 8 weeks of modelling. Based on this, the effects on levels Creative Commons Attribution-NonCommercial 3.0 Unported Licence. of AD-related enzymes and eight neurotransmitters were analyzed. And plasma metabolomics analysis based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to research the metabolic disturbances in the process of AD rat. The results shown the injury on the spatial learning ability of AD rats was gradually aggravated within 4 weeks, reached the maximum at 4 weeks and then was stable until 8 weeks. During 8 weeks of modeling, the levels of enzymes including b-secretase, g-secretase, glycogen synthase kinase-3b (GSK- 3b), acetyl cholinesterase (AchE) and nitric oxide synthase (NOS) were significant increased in the plasma of AD rats.
    [Show full text]
  • (Title of the Thesis)*
    Dionysian Semiotics: Myco-Dendrolatry and Other Shamanic Motifs in the Myths and Rituals of the Phrygian Mother by Daniel Attrell A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Arts in Ancient Mediterranean Cultures Waterloo, Ontario, Canada, 2013 © Daniel Attrell 2013 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The administration of initiation rites by an ecstatic specialist, now known to western scholarship by the general designation of ‗shaman‘, has proven to be one of humanity‘s oldest, most widespread, and continuous magico-religious traditions. At the heart of their initiatory rituals lay an ordeal – a metaphysical journey - almost ubiquitously brought on by the effects of a life-changing hallucinogenic drug experience. To guide their initiates, these shaman worked with a repertoire of locally acquired instruments, costumes, dances, and ecstasy-inducing substances. Among past Mediterranean cultures, Semitic and Indo-European, these sorts of initiation rites were vital to society‘s spiritual well-being. It was, however, the mystery schools of antiquity – organizations founded upon conserving the secrets of plant-lore, astrology, theurgy and mystical philosophy – which satisfied the role of the shaman in Greco-Roman society. The rites they delivered to the common individual were a form of ritualized ecstasy and they provided an orderly context for religiously-oriented intoxication.
    [Show full text]
  • 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
    [Show full text]