Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2011
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 126 S. Ill., graph. Darst. |
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Datensatz im Suchindex
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| adam_text | IMAGE 1
CONTENTS
1 INTRODUCTION 14
1.1 REGENERATION 14
1.1.1 GENERAL MECHANISMS TO PROVIDE PROLIFERATING PROGENITOR CELLS
DURING AMPHIBIAN REGENERATION 14
1.1.2 REGENERATION OF THE AMPHIBIAN LIMB 16
1.1.3 REGENERATION OF AMPHIBIAN SKELETAL MUSCLE 16
1.1.4 NEWT MYOTUBES RE-ENTER THE CELL CYCLE UPON EXPOSURE TO ELEVATED
LEVELS OF SERUM 18
1.1.5 MOUSE MYOTUBES ARE CAPABLE TO DEDIFFERENTIATE IN VITRO 19
1.2 TOWARDS THE IDENTIFICATION O F SPRF 19
1.2.1 CHARACTERIZATION AND PURIFICATION OF SPRF 20
1.2.2 IDENTIFICATION AND EVALUATION O F CANDIDATES 21
1.2.3 BONE MORPHOGENIC PROTEINS 22
1.2.4 NOGGIN - A POTENT AND SPECIFIC INHIBITOR OF BMP SIGNALING . . . .
25 1.3 GOAL O F THIS PHD THESIS 26
2 RESULTS 27
2.1 THE S-PHASE RE-ENTRY ACTIVITY IS RESISTANT TO FREEZING 27
2.2 SERUM BMPS INDUCE S-PHASE RE-ENTRY IN NEWT A1 MYOTUBES 29
2.2.1 RECOMBINANT BOVINE BMPS INDUCE S-PHASE RE-ENTRY O F NEWT A1
MYOTUBES 29
2.2.2 SERUM BMP4 CORRELATES WITH THE S-PHASE RE-ENTRY ACTIVITY . . . 34
2.2.2.1 SERUM BMP4 AND THE SERUM ACTIVITY CORRELATE ALONG THE
PURIFICATION 34
2.2.2.2 SERUM BMP4 AND THE SERUM ACTIVITY CO-FRACTIONATE ALONG SINGLE
STEPS O F THE PURIFICATION 36
2.2.3 RECOMBINANT AND SERUM DERIVED BMP4 DISPLAY DIFFERENCES IN
MOLECULAR WEIGHT 40
2.2.4 RECOMBINANT BMPS ARE LESS ACTIVE THAN SERUM DERIVED BMPS . . 40
2.2.5 SERUM BMPS ARE NECESSARY TO INDUCE S-PHASE RE-ENTRY IN NEWT A1
MYOTUBES 44
2.2.6 EVALUATION OF RECOMBINANT NOGGIN PREPARATIONS FOR THE PULL DOWN
EXPERIMENT 46
2.2.6.1 RECOMBINANT NOGGIN EXPRESSED IN BACTERIA IS LESS AC TIVE THAN
HEK293 CELL NOGGIN 46
2.2.6.2 RECOMBINANT NOGGIN-FC OVER EXPRESSED IN HEK293 CELLS IS A USEFUL
AGENT TO DEPLETE BMPS FROM RECOMBI NANT AS WELL AS SERUM DERIVED
SAMPLES 51
2.2.6.2.1 THE DEPLETION O F SERUM BMPS LEADS TO THE DEPLETION OF THE
S-PHASE RE-ENTRY ACTIVITY . . 52 2.2.6.3 CROSS-LINKING O F NOGGIN-FC TO
PROTEIN G BEADS . . . . 52
2.2.6.4 NOGGIN-FC THAT IS COVALENTLY CROSS-LINKED TO PROTEIN G BEADS IS
STILL BIOLOGICALLY ACTIVE 54
6
HTTP://D-NB.INFO/1029607060
IMAGE 2
2.2.7 SERUM BMPS ARE SUFFICIENT TO INDUCE S-PHASE RE-ENTRY IN A1 MY
OTUBES 57
2.2.7.1 DEPLETION O F BMP4 DEPLETES THE SERUM ACTIVITY . . . . 60
2.3 BMPS ARE ACTIVATED BY SERUM PROTEASES 63
2.3.1 QUALITY MEASUREMENTS O F COMMERCIALLY AVAILABLE THROMBIN AND
PLASMIN PREPARATIONS 63
2.3.2 THROMBIN ENHANCES THE POTENCY OF HUMAN BMP4 64
2.3.3 PLASMIN ENHANCES THE POTENCY O F HUMAN BMPS 66
2.3.4 PLASMIN ENHANCES THE POTENCY OF BMPS FROM DIFFERENT SPECIES . 69
2.3.5 THE PROTEOLYTIC ACTIVATION OF BMPS DOES ACCOUNT FOR THE DISCREP
ANCY IN POTENCY BETWEEN RECOMBINANT AND SERUM-DERIVED BMPS 71 2.3.6
PLASMIN ENHANCES THE POTENCY OF RECOMBINANT BMPS EXPRESSED
IN E. COLI 73
2.4 IDENTIFICATION OF POTENTIAL PLASMIN CLEAVAGE SITES 77
2.4.1 IDENTIFICATION O F CONSERVED RESIDUES IN BMPS 77
2.4.2 IDENTIFICATION OF BMP PEPTIDES BY MASS SPECTROMETRY 78
2.4.3 MUTATIONAL ANALYSIS O F POTENTIAL PLASMIN CLEAVAGE SITES IN HBMP4
82 2.4.3.1 FURIN CO-EXPRESSION INCREASES THE AMOUNT OF MATURE MUTANT
BMP4 PROTEIN 82
2.4.3.2 THE POTENCY OF THE AN-HBMP4 MUTANT IS ENHANCED UPON TREATMENT
WITH THROMBIN AND PLASMIN 85
2.4.3.3 A SINGLE CLEAVAGE AT THE N-TERMINUS DOES NOT ACCOUNT FOR THE
SIZE SHIFT CAUSED DUE TO PLASMIN TREATMENT . . . 86 2.4.3.4
MODIFICATIONS AT THE C-TERMINUS OF BMP4 LEAD TO BIO LOGICAL INACTIVE
PROTEIN 87
2.4.3.5 PLASMIN AND THROMBIN MOST LIKELY TARGET THE LYSINE RESIDUE
LOCATED IN THE CENTRAL REGION O F BMP4 89
3 DISCUSSION 94
3.1 MOLECULAR IDENTIFICATION O F THE SERUM ACTIVITY 94
3.2 CLOTTING PROTEASES ENHANCE THE POTENCY OF BMPS 96
3.3 IDENTIFICATION OF POTENTIAL CLEAVAGE POSITIONS 97
3.4 BMP LEVELS IN PLASMA 98
3.5 INITIATION O F REGENERATION AT THE SITE O F INJURY 98
3.6 OPEN QUESTIONS AND FURTHER PERSPECTIVES 99
4 MATERIALS AND METHODS 102
4.1 BIOCHEMISTRY 102
4.1.1 CHROMATOGRAPHY AND DESALTING 102
4.1.1.1 CRUDE BOVINE THROMBIN 102
4.1.1.2 CATION EXCHANGE CHROMATOGRAPHY 102
4.1.1.3 HYDROPHOBIC INTERACTION CHROMATOGRAPHY 102
4.1.1.4 HEPARIN AFFINITY CHROMATOGRAPHY 102
4.1.1.5 DESALTING ON NAP-5 COLUMNS 103
4.1.1.6 CONCENTRATING AND DESALTING O F DILUTED PROTEIN SAMPLES 103
7
IMAGE 3
4.1.1.7 CAP DIALYSIS 103
4.1.1.8 TUBE DIALYSIS 103
4.1.2 CROSS LINKING OF PROTEINS TO PROTEIN G BEADS 103
4.1.3 IMMUNOPRECIPITATION 103
4.1.3.1 IMMUNOPRECIPITATION OF BMPS USING NOGGIN-FC . . . . 104 4.1.3.2
IMMUNOPRECIPITATION OF BMP4 USING AN ANTI-BMP4 ANTIBODY 104
4.1.4 PROTEOLYTIC ACTIVATION BY SERUM PROTEASES 104
4.1.5 PROTEOLYTIC ACTIVATION BY 3C PROTEASES 104
4.1.6 CHROMOGENIC ASSAYS 105
4.1.6.1 THROMBIN ACTIVITY ASSAY 105
4.1.6.2 PLASMIN ACTIVITY ASSAY 105
4.1.7 INHIBITION OF PROTEASE ACTIVITY 105
4.1.8 BMP INHIBITION 105
4.1.9 PREPARATIVE SDS-PAGE 106
4.1.10 ANALYTICAL SDS-PAGE 106
4.1.11 WESTERN BLOTTING 106
4.1.12 COOMASSIE STAINING OF PROTEIN GELS 106
4.1.13 SILVER STAINING 106
4.1.14 B C A - ASSAY 107
4.1.15 PRECIPITATION OF PROTEINS USING TRICHLOROACETIC ACID (TCA) . . .
107 4.1.16 DEGLYCOSYLATION 107
4.2 CLONING EXPRESSION CONSTRUCTS 107
4.2.1 CONSTRUCTS FOR EXPRESSION IN HEK293 CELL SYSTEM 108
4.2.2 CONSTRUCTS FOR EXPRESSION IN E. COLI 108
4.3 CELL CULTURE 109
4.3.1 A1 MYOBLASTS - PASSAGING 109
4.3.2 A1 MYOTUBE ASSAY 109
4.3.2.1 SETTING UP MYOTUBE ASSAY 109
4.3.2.2 FIXATION AND STAINING O F THE MYOTUBE ASSAY 110
4.3.2.3 COUNTING OF THE MYOTUBE ASSAY 110
4.3.3 EXPRESSION OF RECOMBINANT PROTEINS IN HEK293 CELLS 110
4.3.4 EXPRESSION O F RECOMBINANT BMPS IN E.COLI I L L
4.3.5 EXPRESSION AND IN VITRO REFOLDING OF NOGGIN-AVI I L L
4.3.6 IN VITRO BIOTINYLATION OF NOGGIN-AVI I L L
4.4 MASS SPECTROMETRY I L L
4.4.1 MS ANALYSIS O F BOVINE SERUM DERIVED SAMPLES I L L
4.4.2 MS ANALYSIS O F RECOMBINANT BMPS EXPRESSED IN BACTERIA . . . . 112
5 ACKNOWLEDGMENTS 114
8
|
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| author | Wagner, Ines 1975- |
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| author_facet | Wagner, Ines 1975- |
| author_role | aut |
| author_sort | Wagner, Ines 1975- |
| author_variant | i w iw |
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| bvnumber | BV040643986 |
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| dewey-full | 573.7538893741785 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 573 - Specific physiological systems in animals |
| dewey-raw | 573.7538893741785 |
| dewey-search | 573.7538893741785 |
| dewey-sort | 3573.7538893741785 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Wagner, Ines 1975- Verfasser (DE-588)1027543464 aut Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells von Ines Wagner 2011 126 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2011 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025470991&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wagner, Ines 1975- Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells |
| subject_GND | (DE-588)4113937-9 |
| title | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells |
| title_auth | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells |
| title_exact_search | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells |
| title_full | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells von Ines Wagner |
| title_fullStr | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells von Ines Wagner |
| title_full_unstemmed | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells von Ines Wagner |
| title_short | Identification and proteolytic activation of a serum factor that induces S-phase re-entry in salamander muscle cells |
| title_sort | identification and proteolytic activation of a serum factor that induces s phase re entry in salamander muscle cells |
| topic_facet | Hochschulschrift |
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