Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
|
| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-24578 Inhaltsverzeichnis |
| Beschreibung: | XII, 118 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase |c vorgelegt von Janina Hamberger |
| 264 | 1 | |c 2012 | |
| 300 | |a XII, 118 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Regensburg, Univ., Diss., 2012 | ||
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| 650 | 0 | 7 | |a Streptococcus pneumoniae |0 (DE-588)4244508-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Säugetiere |0 (DE-588)4051253-8 |2 gnd |9 rswk-swf |
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| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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Datensatz im Suchindex
| _version_ | 1804150411217600512 |
|---|---|
| adam_text | IMAGE 1
CONTENTS
1 GENERAL INTRODUCTION 1
1.1. HYALURONAN 2
1.1.1. STRUCTURE AND PHYSICOCHEMICAL PROPERTIES 2
1.1.2. OCCURRENCE, SYNTHESIS AND PHYSIOLOGICAL ROLES 3
1.2. HYALURONAN RECEPTORS AND SIGNALLING 4
1.3. HYALURONAN TURNOVER 6
1.3.1. HYALURONIDASES 6
1.3.1.1. BACTERIAL HYALURONIDASES 6
1.3.1.2. HUMAN HYALURONIDASES 7
1.3.2. MODEL O F HYALURONAN TURNOVER 8
1.4. SIZE-DEPENDENT FUNCTIONS O F HYALURONAN FRAGMENTS 9
.? *
1.5. REFERENCES 10
2 SCOPE AND OBJECTIVES 15
3 EXPRESSION, PURIFICATION AND CHARACTERIZATION O F RECOMBINANT HUMAN
HYAL-2 17
3.1. INTRODUCTION 18
3.2. MATERIALS AND METHODS 20
3.2.1. CULTIVATION AND STORAGE OF STABLY TRANSFECTED
DS-2/PMTHYGRO/HYAL-2 CELLS 20
3.2.2. EXPRESSION OF RHHYAL-2 IN DS-2 CELLS 20
3.2.3. ISOLATION AND PURIFICATION OF RHHYAL-2 BY NI-IMAC 20
3.2.4. BICINCHONINIC ACID (BCA) PROTEIN ASSAY 21
3.2.5. SIMPLIFIED PURIFICATION PROTOCOL FOR RHHYAL-2 21
3.2.6. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 22
3.2.7. WESTERN BLOT ANALYSIS 23
3.2.8. VISCOSIMETRIC HYALURONIDASE ACTIVITY ASSAY 23
3.2.9. POLYACRYLAMIDE GEL ELECTROPHORESIS FOLLOWED BY COMBINED ALCIAN
BLUE SILVER
STAINING 24
3.3. RESULTS AND DISCUSSION 25
3.3.1. PURIFICATION OF RHHYAL-2 FROM DS-2 CELL MEDIUM 25
3.3.2. PURIFICATION OF RHHYAL-2 ACCORDING TO A SIMPLIFIED PROTOCOL USING
NI
SEPHAROSE* 6 FF 26
V
HTTP://D-NB.INFO/1037894219
IMAGE 2
3.3.3. DETERMINATION OF ENZYMATIC ACTIVITY O F PURIFIED RHHYAL-2 BY
VISCOSIMETRY_27
3.3.4. DETERMINATION OF ENZYMATIC ACTIVITY O F RHHYAL-2 BY
ELECTROPHORESIS 29
3.4. SUMMARY AND CONCLUSIONS 30
3.5. REFERENCES 31
4 ENZYMATIC ACTIVITY O F HYAL-2 IN BLOOD PLATELETS 35
4.1. INTRODUCTION 36
4.2. MATERIALS AND METHODS 38
4.2.1. ISOLATION OF HUMAN PLATELETS ; 38
4.2.2. ISOLATION O F MURINE PLATELETS 38
4.2.3. PREPARATION OF HUMAN PLATELET MEMBRANES 38
I 4.2.4. SDS-PAGE AND WESTERN BLOT 39
4.2.5. 2-D GEL ELECTROPHORESIS 39
4.2.6. GEL PERMEATION CHROMATOGRAPHY (GPC) 40
4.2.7. VISCOSIMETRIC AND ELECTROPHORETIC HYALURONIDASE ACTIVITY ASSAYS
40
4.2.8. ZYMOGRAPHY 40
4.2.9. ACTIVATION OF PLATELETS 41
4.2.10. CA 2+ ASSAY FOR MEASURING PLATELET ACTIVATION 41
4.3. RESULTS AND DISCUSSION 42
4.3.1. SDS-PAGE AND WESTERN BLOT ANALYSIS O F HYAL-2 IN HUMAN PLATELETS
42
4.3.2. 2-D GEL ELECTROPHORESIS 43
4.3.3. DETECTION OF ENZYMATIC ACTIVITY O F HYAL-2 IN NON-ACTIVATED HUMAN
PLATELETS BY
VISCOSIMETRY 44
4.3.4. DEPENDENCY OF SUBSTRATE DEGRADATION BY PLATELET-DERIVED HYAL-2 ON
THE SOURCE
OF.HA 47
4.3.5. GEL PERMEATION CHROMATOGRAPHY FOR DETERMINATION OF HYALURONAN
FRAGMENTS
GENERATED BY PLATELET-ASSOCIATED HYAL-2 48
4.3.6. DETERMINATION OF ENZYMATIC ACTIVITY O F HYAL-2 IN NON-ACTIVATED
HUMAN
PLATELETS BY ELECTROPHORESIS 49
4.3.7. ZYMOGRAPHY 50
4.3.8. ACTIVATION OF HUMAN PLATELETS 51
4.3.9. HYALURONAN DEGRADATION BY HYAL-2 IN ACTIVATED HUMAN PLATELETS 52
4.3.10. DETECTION OF ENZYMATIC ACTIVITY OF HYAL-2 IN MURINE PLATELETS BY
VISCOSIMETRY 53
VI
IMAGE 3
4.3.11. DETECTION OF HYALURONAN FRAGMENTS GENERATED BY MURINE
PLATELET-DERIVED
HYAL-2 USING ELECTROPHORESIS_ 54
4.3.12. HYALURONAN DEGRADATION BY HYAL-2 DERIVED FROM ACTIVATED MURINE
PLATELETS 55
4.4. SUMMARY AND CONCLUSION 56
4.5. REFERENCES 58
5 DETERMINATION O F HYAL-2 ACTIVITY IN RED BLOOD CELLS 63
5.1. INTRODUCTION M
5.2. MATERIALS AND METHODS : 65
5.2.1. ISOLATION OF HUMAN AND MURINE RED BLOOD CELLS 65
5.2.2. DETERMINATION OF RBC NUMBER IN MURINE BLOOD 65
5.2.3. PREPARATION OF ERYTHROCYTE GHOJT MEMBRANES 65
5.2.4. SDS-PAGE AND WESTERN BLOT ANALYSIS 65
5.2.5. VISCOSIMETRY AND PAGE FOR DETERMINATION OF HYAL-2 ACTIVITY IN RBC
66
5.3. RESULTS AND DISCUSSION 66
5.3.1. IDENTIFICATION OF HYAL-2 IN RBCS AND RBC MEMBRANES BY IMMUNO
DETECTION 66
5.3.2. HYALURONIDASE ACTIVITY ASSAYS O F HYAL-2 IN RBC 66
5.3.3. HYALURONIDASE ACTIVITY ASSAY OF HYAL-2 IN RBC MEMBRANES 67
5.4. SUMMARY AND CONCLUSION 68
5.5. REFERENCES 69
6 INVESTIGATIONS ON THE EFFECT O F HYALURONAN DIGESTION MIXTURES ON THE
PROLIFERATION O F HUMAN ENDOTHELIAL CELLS 71
6.1. INTRODUCTION 72
6.2. MATERIALS AND METHODS _ 7 3
6.2.1. CELL CULTURE CONDITIONS AND STORAGE 73
6.2.2. PREPARATION OF CELL LYSATES FOR SDS-PAGEAVESTERN BLOT 73
6.2.3. SDS-PAGE AND WESTERN BLOT ANALYSIS 73
6.2.4. HYALURONAN RECEPTOR DETECTION BY FLOW CYTOMETRY 74
6.2.5. PREPARATION OF DIFFERENT HYALURONAN DIGESTION MIXTURES 74
6.2.6. CRYSTAL VIOLET ASSAY FOR PROLIFERATION STUDIES 75
6.3. RESULTS AND DISCUSSION _ 7 5
VLL
IMAGE 4
6.3.1. EXPRESSION OF THE HYALURONAN RECEPTORS CD44 AND RHAMM BY HMEC-1
CELLS 75
6.3.1.1. WESTERN BLOT ANALYSIS 75
6.3.1.2. DETERMINATION O F CD44 AND RHAMM IN ENDOTHELIAL CELLS BY FLOW
CYTO
METRY 76
6.3.2. CHARACTERIZATION OF THE DIFFERENT HYALURONAN DIGESTS 77
6.3.3. INVESTIGATIONS OF THE EFFECT O F HA DIGESTS ON THE PROLIFERATION
O F HUMAN
ENDOTHELIAL CELLS _ 7 9
6.4. SUMMARY AND CONCLUSION ; 80
6.5. REFERENCES 81
7 PURIFICATION O F STREPTOCOCCUS PNEUMONIAE HYALURONATE LYASE AND J
SCREENING FOR POSSIBLE SMALL MOLECULE INHIBITORS 85
7.1. /I NTROD UCTION 86
7.2. MATERIALS AND METHODS 88
7.2.1. SMALL SCALE EXPRESSION O F S. PNEUMONIAE HYALURONATE LYASE 88
7.2.2. SDS-PAGE AND WESTERN BLOT ANALYSIS 88
7.2.3. PURIFICATION OF 5PHYL WITH NI-SEPHAROSE* 6 FF 88
7.2.4. COLORIMETRIC HYALURONIDASE ACTIVITY ASSAY 89
7.2.5. TURBIDIMETRIC HYALURONIDASE ACTIVITY ASSAY FOR SCREENING 90
7.3. RESULTS AND DISCUSSION 91
7.3.1. SMALL SCALE EXPRESSION O F S. PNEUMONIAE HYALURONATE LYASE 91
7.3.2. PURIFICATION AND CHARACTERIZATION O F SPNHYL AFTER LARGE SCALE
EXPRESSION 92
7.3.3. SCREENING OF SMALL MOLECULES FOR INHIBITION OF SPNHYL 92
7.4. SUMMARY AND CONCLUSION 100
7.5. REFERENCES 101
8 SUMMARY 103
APPENDIX: IN VITRO INVESTIGATIONS ON LOBAPLATIN AGAINST TRIPLE-NEGATIVE
HUMAN BREAST CANCER CELLS 107
1 INTRODUCTION 108
2 MATERIALS AND METHODS 109
2.1 PLATINUM DRUGS 109
VIII
IMAGE 5
2.2 CELL LINES AND CULTURE CONDITIONS 109
2.3 SUBCUTANEOUS INJECTION OF HCC-1806 AND -1937 CELLS AND HISTOLOGY 109
2.4 CHEMOSENSITIVITY ASSAY BASED ON CRYSTAL VIOLET STAINING 110
3 RESULTS AND DISCUSSION 111
3.1 CHEMOSENSITIVITY O F TRIPLE NEGATIVE BREAST CANCER CELLS 111
3.2 TUMORIGENICITY AND HISTOLOGY OF HCC-1806 AND HCC-1937 TUMORS 114
4 SUMMARY 116
5 REFERENCES 116
IX
|
| any_adam_object | 1 |
| author | Hamberger, Janina |
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| author_role | aut |
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| dewey-raw | 610 570 |
| dewey-search | 610 570 |
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| discipline | Chemie / Pharmazie Biologie Medizin |
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| publishDateSort | 2012 |
| record_format | marc |
| spelling | Hamberger, Janina Verfasser aut Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase vorgelegt von Janina Hamberger 2012 XII, 118 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2012 Hyaluronidasen (DE-588)4204239-2 gnd rswk-swf Enzyminhibitor (DE-588)4152479-2 gnd rswk-swf Virulenzfaktor (DE-588)4354782-5 gnd rswk-swf Hyaluronsäure (DE-588)4160834-3 gnd rswk-swf Streptococcus pneumoniae (DE-588)4244508-5 gnd rswk-swf Säugetiere (DE-588)4051253-8 gnd rswk-swf Enzymaktivität (DE-588)4152470-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Hyaluronidasen (DE-588)4204239-2 s Enzyminhibitor (DE-588)4152479-2 s Virulenzfaktor (DE-588)4354782-5 s Streptococcus pneumoniae (DE-588)4244508-5 s DE-604 Säugetiere (DE-588)4051253-8 s Hyaluronsäure (DE-588)4160834-3 s Enzymaktivität (DE-588)4152470-6 s Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-24578 http://epub.uni-regensburg.de/24578/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-24578 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026024201&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hamberger, Janina Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase Hyaluronidasen (DE-588)4204239-2 gnd Enzyminhibitor (DE-588)4152479-2 gnd Virulenzfaktor (DE-588)4354782-5 gnd Hyaluronsäure (DE-588)4160834-3 gnd Streptococcus pneumoniae (DE-588)4244508-5 gnd Säugetiere (DE-588)4051253-8 gnd Enzymaktivität (DE-588)4152470-6 gnd |
| subject_GND | (DE-588)4204239-2 (DE-588)4152479-2 (DE-588)4354782-5 (DE-588)4160834-3 (DE-588)4244508-5 (DE-588)4051253-8 (DE-588)4152470-6 (DE-588)4113937-9 |
| title | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase |
| title_auth | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase |
| title_exact_search | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase |
| title_full | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase vorgelegt von Janina Hamberger |
| title_fullStr | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase vorgelegt von Janina Hamberger |
| title_full_unstemmed | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase vorgelegt von Janina Hamberger |
| title_short | Characterization of mammalian hyaluronidase-2 activity and identification of inhibitors of Streptococcal hyaluronan lyase |
| title_sort | characterization of mammalian hyaluronidase 2 activity and identification of inhibitors of streptococcal hyaluronan lyase |
| topic | Hyaluronidasen (DE-588)4204239-2 gnd Enzyminhibitor (DE-588)4152479-2 gnd Virulenzfaktor (DE-588)4354782-5 gnd Hyaluronsäure (DE-588)4160834-3 gnd Streptococcus pneumoniae (DE-588)4244508-5 gnd Säugetiere (DE-588)4051253-8 gnd Enzymaktivität (DE-588)4152470-6 gnd |
| topic_facet | Hyaluronidasen Enzyminhibitor Virulenzfaktor Hyaluronsäure Streptococcus pneumoniae Säugetiere Enzymaktivität Hochschulschrift |
| url | http://epub.uni-regensburg.de/24578/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-24578 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026024201&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hambergerjanina characterizationofmammalianhyaluronidase2activityandidentificationofinhibitorsofstreptococcalhyaluronanlyase |