DOCSLIB.ORG

(12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. The invention further relates to drug-Screening methods using U.S. PATENT DOCUMENTS the protease polypeptides and polynucleotides to identify 5,552,526 A 9/1996 Nakamura et all 530/350 agonists and antagonists for diagnosis and treatment. The 5883.241 A * 3/1999 Docherty et al. r 536/23.2 invention further encompasses agonists and antagonists 5.990.293 A * 11/1999 Docherty et al. .......... 536/23.1 based on the protease polypeptides and polynucleotides. The invention further relates to procedures for producing the FOREIGN PATENT DOCUMENTS protease polypeptides and polynucleotides. EP O874050 A2 * 10/1998 ........... C12N/15/15 WO WO-98/55643 A1 * 12/1998 ........... C12P/21/02 1 Claim, No Drawings US 6,395,889 B1 1 2 NUCLEIC ACID MOLECULES ENCODING The invention also provides an isolated fragment or HUMAN PROTEASE HOMOLOGS portion of any of SEQ ID NOS: 1-33 and the complement of SEQ ID NOS: 1-33. In preferred embodiments, the FIELD OF THE INVENTION fragment is useful as a probe or primer, and/or is at least 15, The invention relates to newly identified polynucleotides more preferably at least 18, even more preferably 20–25, 30, having homology to various protease families. The inven 50, 100, 200 or more nucleotides in length. tion also relates to protease polypeptides. The invention The invention also provides isolated fragments of the further relates to methods using the protease polypeptides polypeptides. and polynucleotides as a target for diagnosis and treatment In another embodiment, the invention provides an isolated in protease-mediated and related disorders. The invention nucleic acid molecule that hybridizes under high Stringency further relates to drug-Screening methods using the protease conditions to a nucleotide Sequence Selected from the group polypeptides and polynucleotides to identify agonists and consisting of SEQ ID NOS: 1-33 and the complements of antagonists for diagnosis and treatment. The invention fur SEO ID NOS: 1-33. ther encompasses agonists and antagonists based on the The invention further provides nucleic acid constructs protease polypeptides and polynucleotides. The invention 15 comprising the nucleic acid molecules described above. In a further relates to procedures for producing the protease preferred embodiment, the nucleic acid molecules of the polypeptides and polynucleotides. invention are operatively linked to a regulatory Sequence. The invention also provides vectors and host cells for BACKGROUND OF THE INVENTION expressing the protease nucleic acid molecules and polypep Proteases are a major target for drug action and develop tides and particularly recombinant vectors and host cells. ment. Accordingly, it is valuable to the field of pharmaceu The invention also provides methods of making the tical development to identify and characterize previously vectors and host cells and methods for using them to produce unknown protease nucleic acids and polypeptides. The the protease nucleic acid molecules and polypeptides. present invention advances the State of the art by providing 25 The invention also provides antibodies or antigen-binding previously unidentified human protease Sequences. fragments thereof that Selectively bind the protease polypep tides and fragments. SUMMARY OF THE INVENTION The invention also provides methods of Screening for It is an object of the invention to identify novel proteases. compounds that modulate expression or activity of the It is a further object of the invention to provide novel protease polypeptides or nucleic acid (RNA or DNA). protease polypeptides that are useful as reagents or targets in The invention also provides a process for modulating protease assays applicable to treatment and diagnosis of protease polypeptide or nucleic acid expression or activity, protease-mediated disorders. especially using the Screened compounds. Modulation may It is a further object of the invention to provide poly 35 be used to treat conditions related to aberrant activity or nucleotides corresponding to the novel protease polypep expression of the protease polypeptides or nucleic acids. tides that are useful as targets and reagents in protease assays The invention also provides assays for determining the applicable to treatment and diagnosis of protease-mediated presence or absence of and level of the protease polypep disorders and useful for producing novel protease polypep tides or nucleic acid molecules in a biological Sample, tides by recombinant methods. 40 including for disease diagnosis. Aspecific object of the invention is to identify compounds The invention also provides assays for determining the that act as agonists and antagonists and modulate the expres presence of a mutation in the protease polypeptides or Sion of the novel proteases. nucleic acid molecules, including for disease diagnosis. A further specific object of the invention is to provide In still a further embodiment, the invention provides a compounds that modulate expression of the proteases for 45 computer readable means containing the nucleotide and/or treatment and diagnosis of protease- related disorders. amino acid Sequences of the nucleic acids and polypeptides The present invention is based on the identification of of the invention, respectively. novel nucleic acid molecules that are homologous to pro DETAILED DESCRIPTION OF THE tease Sequences. INVENTION 50 Thus, in one aspect, the invention provides an isolated The following text describes classes of proteins with nucleic acid molecule that comprises a nucleotide Sequence which the Sequences described herein have been compared selected from the group consisting of SEQ ID NOS: 1-33 with respect to homology. and the complements of SEQ ID NOS: 1-33. Eukaryotic and Viral Aspartyl Active Sites In another aspect, the invention provides isolated proteins 55 Aspartyl proteases, also known as acid proteases, (EC and polypeptides encoded by nucleic acid molecules of the 3.4.23.-), are a widely distributed family of proteolytic invention. enzymes (Foltman (1981) Essays Biochem 17:52–84; Davis In another embodiment, the invention provides an isolated (1990) Annu. Rev. Biophys. Chem. 19:189–215; Rao et al. nucleic acid molecule that comprises a nucleotide Sequence (1991) Biochemistry 30: 4663-4671) that exist in that is at least about 60% identical, preferably at least about 60 vertebrates, fungi, plants, retroviruses and Some plant 80% identical, preferably at least about 85% identical, more Viruses. ASpartate proteases of eukaryotes are monomeric preferably at least about 90% identical, and even more enzymes which consist of two domains. Each domain con preferably at least about 95% identical, and most preferably tains an active site centered on a catalytic aspartyl residue. about 98% or more identical to a nucleotide sequence The two domains most probably evolved from the duplica selected from the group consisting of SEQ ID NOS: 1-33 65 tion of an ancestral gene encoding a primordial domain. and the complements of SEQ ID NOS: 1-33. Currently known eukaryotic aspartyl proteases include, but The invention also provides isolated variant polypeptides. are not limited to: US 6,395,889 B1 3 4 Vertebrate gastric pepsins A and C (also known as Eukaryotic Thiol (Cysteine) Proteases Active Sites Eukary gastricsin). otic thiol proteases (EC 3.4.22.-) (Dufour (1988) Biochimie Vertebrate chymosin (rennin), involved in digestion and 70: 1335–1342) are a family of proteolytic enzymes which used for making cheese. contain an active Site cysteine. Catalysis proceeds through a Vertebrate lysosomal cathepsins D (EC 3.4.23.5) and E thioester intermediate and is facilitated by a nearby histidine (EC 3.4.23.34). Side chain; an asparagine completes the essential catalytic Mammalian renin (EC 3.4.23.15) whose function is to triad. Proteases that belong to this family include, but are not generate angiotensin I from angiotensinogen in the limited to: plasma. Vertebrate lysosomal cathepsins B (EC 3.4.22. 1), H (EC Fungal proteases Such as aspergillopepsin A (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27) 3.4.23.18), candidapepsin (EC 3.4.23.24), mucoropep (Kirschke et al. (1995) Protein Prof. 2:1587–1643). sin (EC 3.4.23.23) (mucor rennin), endothiapepsin (EC Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) 3.4.23.22), polyporopepsin (EC 3.4.23.29), and rhizo (also known as cathepsin C) (Kirschke et al.
Load more

Recommended publications
  • Cross-Linking of Alpha 2-Plasmin Inhibitor to Fibrin by Fibrin- Stabilizing Factor
    Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin- stabilizing factor. Y Sakata, N Aoki J Clin Invest. 1980;65(2):290-297. https://doi.org/10.1172/JCI109671. Research Article The concentration of alpha 2-plasmin inhibitor in blood plasma is higher than that in serum obtained from the blood clotted in the presence of calcium ions, but is the same as that in serum obtained in the absence of calcium ions. Radiolabeled alpha2-plasmin inhibitor was covalently bound to fibrin only when calcium ions were present at the time of clotting of plasma or fibrinogen. Whereas, when batroxobin, a snake venom enzyme that lacks the ability to activate fibrin- stabilizing factor, was used for clotting fibrinogen, the binding was not observed. When fibrin-stablizing, factor-deficient plasma was clotted, the specific binding of alpha 2-plasmin inhibitor to fibrin did not occur even in the presence of calcium ions and the concentration of alpha 2-plasmin inhibitor in serum was the same as that in plasma. Monodansyl cadaverine, a fluorescent substrate of the fibrin-stablizing factor, was incorporated into alpha 2-plasmin inhibitor by activated fibrin-stablizing factor. All these findings indicate that alpha 2-plasmin inhibitor is cross-linked to fibrin by activated fibrin-stabilizing factor when blood is clotted. Analysis of alpha 2-plasmin inhibitor-incorporated fibrin by sodium dodecyl sulfate gel electrophoresis showed that the inhibitor was mainly cross-linked to polymerized alpha-chains of cross-linked fibrin. Cross-linking of alpha 2-plasmin inhibitor to fibrin renders fibrin clot less susceptible to fibrinolysis by plasmin.
    [Show 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,
    [Show full text]
  • Structure of Human Aspartyl Aminopeptidase Complexed With
    Chaikuad et al. BMC Structural Biology 2012, 12:14 http://www.biomedcentral.com/1472-6807/12/14 RESEARCH ARTICLE Open Access Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family Apirat Chaikuad1, Ewa S Pilka1, Antonio De Riso2, Frank von Delft1, Kathryn L Kavanagh1, Catherine Vénien-Bryan2, Udo Oppermann1,3 and Wyatt W Yue1* Abstract Backround: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β- hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference.
    [Show full text]
  • Cei'itre for Newfoundi.Ano Studies
    CEI'ITRE FOR NEWFOUNDI.ANO STUDIES TOTAL OF 10 PACES ONLY MAY BE XEROXED (W1thout Author·• Pt-rminton) PURIFICATION AND CHARACTERIZATION OF MAJOR GELATIN­ CLEAVAGE ACTIVITIES IN THE APICALLY LOCATED EXTRACELLULAR MATRIX OF THE SEA URCHIN EMBRYO by Lavanya Ranganathan A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Biochemistry Memorial University of Newfoundland and Labrador May, 2004 St.John's Newfoundland and Labrador Canada Library and Bibliotheque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de !'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre reference ISBN: 0-612-99107-5 Our file Notre reference ISBN: 0-612-99107-5 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a Ia Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I' Internet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve Ia propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these.
    [Show full text]
  • (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.
    [Show full text]
  • A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of Dimiq, a Potent Indolo[2,3- B]Quinoline Agent, Against Candida Albicans Biofilms
    A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of DiMIQ, a Potent Indolo[2,3- b]Quinoline Agent, against Candida albicans Biofilms Robert Zarnowski 1,2*, Anna Jaromin 3*, Agnieszka Zagórska 4, Eddie G. Dominguez 1,2, Katarzyna Sidoryk 5, Jerzy Gubernator 3 and David R. Andes 1,2 1 Department of Medicine, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA; [email protected] (E.G.D.); [email protected] (D.R.A.) 2 Department of Medical Microbiology, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA 3 Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland; [email protected] 4 Department of Medicinal Chemistry, Jagiellonian University Medical College, 30-688 Cracow, Poland; [email protected] 5 Department of Pharmacy, Cosmetic Chemicals and Biotechnology, Team of Chemistry, Łukasiewicz Research Network-Industrial Chemistry Institute, 01-793 Warsaw, Poland; [email protected] * Correspondence: [email protected] (R.Z.); [email protected] (A.J.); Tel.: +1-608-265-8578 (R.Z.); +48-71-3756203 (A.J.) Label-Free Cellular Proteomics of Candida albicans biofilms treated with DiMIQ Identified Proteins Accession # Alternate ID Gene names (ORF ) WT DIMIQ Z SCORE Proteins induced by DiMIQ Arginase (EC 3.5.3.1) A0A1D8PP00 CAR1 CAALFM_C504490CA 0.000 6.648 drug induced Glucan 1,3-beta-glucosidase BGL2 (EC 3.2.1.58) (Exo-1Q5AMT2 BGL2 CAALFM_C402250CA
    [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]
  • Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection
    Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection. Suchita Singh1,+, Rakesh Arya2,3,+, Rhishikesh R Bargaje1, Mrinal Kumar Das2,4, Subia Akram2, Hossain Md. Faruquee2,5, Rajendra Kumar Behera3, Ranjan Kumar Nanda2,*, Anurag Agrawal1 1Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR- Institute of Genomics and Integrative Biology, New Delhi, 110025, India. 2Translational Health Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. 3School of Life Sciences, Sambalpur University, Jyoti Vihar, Sambalpur, Orissa, 768019, India. 4Department of Respiratory Sciences, #211, Maurice Shock Building, University of Leicester, LE1 9HN 5Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia- 7003, Bangladesh. +Contributed equally for this work. S-1 70 G1 S 60 G2/M 50 40 30 % of cells 20 10 0 CURI LPSI LPSCUR Figure S1: Effect of curcumin and/or LPS treatment on A549 cell viability A549 cells were treated with curcumin (10 µM) and/or LPS or 1 µg/ml for the indicated times and after fixation were stained with propidium iodide and Annexin V-FITC. The DNA contents were determined by flow cytometry to calculate percentage of cells present in each phase of the cell cycle (G1, S and G2/M) using Flowing analysis software. S-2 Figure S2: Total proteins identified in all the three experiments and their distribution betwee curcumin and/or LPS treated conditions. The proteins showing differential expressions (log2 fold change≥2) in these experiments were presented in the venn diagram and certain number of proteins are common in all three experiments.
    [Show full text]
  • Correlation of the Production of Plasminogen Activator with Tumor Metastasis in Bi 6 Mouse Melanoma Cell Lines
    [CANCER RESEARCH 40, 288-292, February 1980 0008-5472/80/0040-0000$02.00 Correlation of the Production of Plasminogen Activator with Tumor Metastasis in Bi 6 Mouse Melanoma Cell Lines Bosco Shang Wang,2 Gerard A. McLoughlin,3 Jerome P. Richie, and John A. Mannick Deportment of Surgery, Peter Bent Brigham Hospital (B. S. W., G. A. M., J. P. R., J. A. M.j, and Department of Pathology (B. S. W.j, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT circulating cancer cells were easily detected in patients whose blood had a higher level of FA. The correlation between the production of plasminogen ac Although the FA of a tumor has been suggested as correlat tivator (PA) of tumors and their metastatic potential was stud ing with its metastatic potential, direct and quantitative evi ied. Bi 6 melanoma cells and ‘‘Bi6 mets' ‘cells (harvested from dence to support this hypothesis is lacking. Therefore, we have the pulmonary metastatic nodules of C57BL/6J mice bearing investigated this issue by comparing the FA of the Bi 6 mouse Bi 6 isografts) were examined with respect to their fibrinolytic melanoma and several of its sublines varying in their potential activity (FA) in tissue culture. Bi 6 mets cells had a significantly for forming metastases. higher FA than did Bi 6 cells. Fl (a Bi 6 subline with a lower incidence of metastasis) and Fl 0 (a highly metastatic Bi 6 subline) were also studied. Fl 0 cells produced more FA than MATERIALS AND METHODS did Fi cells. The difference between the FA's of these tumors Tumors.
    [Show full text]
  • Gent Forms of Metalloproteinases in Hydra
    Cell Research (2002); 12(3-4):163-176 http://www.cell-research.com REVIEW Structure, expression, and developmental function of early diver- gent forms of metalloproteinases in Hydra 1 2 3 4 MICHAEL P SARRAS JR , LI YAN , ALEXEY LEONTOVICH , JIN SONG ZHANG 1 Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City, Kansas 66160- 7400, USA 2 Centocor, Malvern, PA 19355, USA 3 Department of Experimental Pathology, Mayo Clinic, Rochester, MN 55904, USA 4 Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA ABSTRACT Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydro- lytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra. Key words: Hydra, metalloproteinases, development, astacin, matrix metalloproteinases, endothelin.
    [Show full text]
  • Penicillopepsin-JT2, a Recombinant Enzyme from Penicillium Janthinellum and the Contribution of a Hydrogen Bond in Subsite S3 to Kcat
    Protein Science ~2000!, 9:991–1001. Cambridge University Press. Printed in the USA. Copyright © 2000 The Protein Society Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to kcat QING-NA CAO,1,3 MARLENE STUBBS,1 KENNY Q.P. NGO,1 MICHAEL WARD,2 ANNIE CUNNINGHAM,1 EMIL F. PAI,1 GUANG-CHOU TU,1,3 and THEO HOFMANN1 1 Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada 2 Genencor International, Inc., 925 Page Mill Road, Palo Alto, California 94304-1013 ~Received August 30, 1999; Final Revision February 7, 2000; Accepted March 10, 2000! Abstract The nucleotide sequence of the gene ~ pepA! of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin ~which we propose to call penicillopepsin-JT1! has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P29 of substrates, are similar to those of penicillopepsin-JT1, endothia- pepsin, rhizopuspepsin, and pig pepsin.
    [Show full text]
  • Proteolytic Enzymes in Grass Pollen and Their Relationship to Allergenic Proteins
    Proteolytic Enzymes in Grass Pollen and their Relationship to Allergenic Proteins By Rohit G. Saldanha A thesis submitted in fulfilment of the requirements for the degree of Masters by Research Faculty of Medicine The University of New South Wales March 2005 TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF FIGURES 6 LIST OF TABLES 8 LIST OF TABLES 8 ABBREVIATIONS 8 ACKNOWLEDGEMENTS 11 PUBLISHED WORK FROM THIS THESIS 12 ABSTRACT 13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES 14 1.1 Defining Asthma and its Clinical Presentation 14 1.2 Inflammatory Responses in Asthma 15 1.2.1 The Early Phase Response 15 1.2.2 The Late Phase Reaction 16 1.3 Effects of Airway Inflammation 16 1.3.1 Respiratory Epithelium 16 1.3.2 Airway Remodelling 17 1.4 Classification of Asthma 18 1.4.1 Extrinsic Asthma 19 1.4.2 Intrinsic Asthma 19 1.5 Prevalence of Asthma 20 1.6 Immunological Sensitisation 22 1.7 Antigen Presentation and development of T cell Responses. 22 1.8 Factors Influencing T cell Activation Responses 25 1.8.1 Co-Stimulatory Interactions 25 1.8.2 Cognate Cellular Interactions 26 1.8.3 Soluble Pro-inflammatory Factors 26 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation 30 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES 33 1 2.1 The Role of Pollen Allergens in Asthma 33 2.2 Environmental Factors influencing Pollen Exposure 33 2.3 Classification of Pollen Sources 35 2.3.1 Taxonomy of Pollen Sources 35 2.3.2 Cross-Reactivity between different Pollen Allergens 40 2.4 Classification of Pollen Allergens 41 2.4.1
    [Show full text]