Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
|
| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-280481 Inhaltsverzeichnis |
| Beschreibung: | XI, 173 S. Ill., graph. Darst. |
Internformat
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| 084 | |a VS 5075 |0 (DE-625)147684: |2 rvk | ||
| 100 | 1 | |a Nordemann, Uwe |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs |c vorgelegt von Uwe Nordemann |
| 246 | 1 | 3 | |a Radioligand binding and reporter gene assays for histamine H3 and H4 receptor species orthologs |
| 264 | 1 | |c 2013 | |
| 300 | |a XI, 173 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Regensburg, Univ., Diss., 2013 | ||
| 650 | 0 | 7 | |a Histaminrezeptor |0 (DE-588)4257968-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Ligand |g Biochemie |0 (DE-588)4606532-5 |2 gnd |9 rswk-swf |
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| 689 | 0 | 0 | |a Histaminrezeptor |0 (DE-588)4257968-5 |D s |
| 689 | 0 | 1 | |a Ligand |g Biochemie |0 (DE-588)4606532-5 |D s |
| 689 | 0 | 2 | |a Isotopenmarkierung |0 (DE-588)4176811-5 |D s |
| 689 | 0 | 3 | |a Bindestelle |0 (DE-588)4145563-0 |D s |
| 689 | 0 | |5 DE-604 | |
| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:355-epub-280481 |
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| 912 | |a ebook | ||
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Datensatz im Suchindex
| _version_ | 1804150260401963008 |
|---|---|
| adam_text | IMAGE 1
V
CONTENTS
CHAPTER 1 GENERAL INTRODUCTION 1
1.1 G-PROTEIN COUPLED RECEPTORS (GPCRS) 2
1.1.1 GPCRS AS DRUG TARGET 2
1.1.2 STRUCTURE AND CLASSIFICATION 2
1.1.3 ADVANCES IN X-RAY CRYSTALLOGRAPHY FOR GPCRS 5
1.1.4 SIGNAL TRANSDUCTION 7
1.1.4.1 G-PROTEIN MEDIATED SIGNAL TRANSDUCTION 7
1.1.4.2 P-ARRESTIN DEPENDENT SIGNALING 8
1.1.4.3 MODELS OF GPCR SIGNALING, LIGAND CLASSIFICATION AND FUNCTIONAL
SELECTIVITY .... 10
1.2 HISTAMINE AND THE HISTAMINE RECEPTOR FAMILY 12
1.2.1 THE BIOGENIC AMINE HISTAMINE 12
1.2.2 HISTAMINE RECEPTORS AND THEIR LIGANDS 15
1.2.2.1 THE HISTAMINE HI RECEPTOR 15
1.2.2.2 THE HISTAMINE H 2 RECEPTOR 17
1.2.2.3 THE HISTAMINE H 3 RECEPTOR 19
1.2.2.4 THE HISTAMINE H 4 RECEPTOR 22
1.3 REFERENCES 26
CHAPTER 2 SCOPE AND OBJECTIVES 39
CHAPTER 3 DEVELOPMENT OF RADIOLIGAND BINDING ASSAYS FOR HUMAN AND MOUSE
HISTAMINE H 4 RECEPTORS 41
3.1 RADIOLIGAND BINDING ASSAY FOR THE HUMAN HISTAMINE H 4 RECEPTOR 42
3.1.1 INTRODUCTION 42
3.1.2 MATERIALS AND METHODS 44
3.1.2.1 CELL CULTURE 44
3.1.2.2 CHEMOSENSITIVITY ASSAY 44
3.1.2.3 H 4 RECEPTOR LIGANDS 45
3.1.2.4 WHOLE CELL RADIOLIGAND BINDING ASSAY 46
3.1.3 RESULTS AND DISCUSSION 49
3.1.3.1 EFFECT OF GENETICIN (G418) ON HEK293T CELLS 49
3.1.3.2 SATURATION BINDING ASSAY 49
3.1.3.3 COMPETITION BINDING ASSAY 50
3.2 RADIOLIGAND BINDING ASSAY FOR THE MOUSE HISTAMINE H 4 RECEPTOR 56
3.2.1 INTRODUCTION 56
HTTP://D-NB.INFO/1037035976
IMAGE 2
VI
3.2.2 MATERIALS AND METHODS 57
3.2.2.1 SUBCLONING OF THE PCDNA3.1(+)SF-MH 4R -HIS6 VECTOR 57
3.2.2.1.1 RESTRICTION ENZYME DIGESTION 57
3.2.2.1.2 AGAROSE GEL ELECTROPHORESIS, GEL EXTRACTION AND DETERMINATION
OF DNA
CONCENTRATION 58
3.2.2.1.3 LIGATION OF DNA FRAGMENTS 59
3.2.2.1.4 PREPARATION OF MEDIA AND AGAR PLATES 59
3.2.2.1.5 TRANSFORMATION OF COMPETENT E. COLI 60
3.2.2.1.6 PREPARATION OF GLYCEROL CULTURES AND PLASMID DNA (MAXI-PREP)
60
3.2.2.1.7 RESTRICTION ANALYSIS AND SEQUENCING OF PCDNA3.1(+)SF-MH
4R-HIS6 61
3.2.2.2 STABLE TRANSFECTION OF HEK293T CELLS WITH THE PCDNA3.1(+)SF-MH
4R- HIS6 VECTOR 62
3.2.2.3 WHOLE CELL RADIOLIGAND BINDING ASSAY 62
3.2.2.4 IMIDAZOLYLCYCLOPENTYLMETHYLCYANOGUANIDINES UR-RG94 AND UR-RG98
63
3.2.3 RESULTS AND DISCUSSION 64
3.2.3.1 SELECTION OF THE TRANSFECTED CELLS 64
3.2.3.2 SATURATION BINDING ASSAYS 65
3.2.3.3 COMPETITION BINDING ASSAY 66
3.3 SUMMARY AND CONCLUSION 70
3.4 REFERENCES 7 1
CHAPTER 4 DEVELOPMENT OF LUMINESCENCE BASED REPORTER GENE ASSAYS FOR THE
HUMAN, MOUSE AND RAT HISTAMINE H 4 RECEPTOR 75
4.1 DEVELOPMENT OF A REPORTER GENE ASSAY FOR THE HUMAN HISTAMINE H 4
RECEPTOR 76
4.1.1 INTRODUCTION 76
4.1.2 MATERIALS AND METHODS 80
4.1.2.1 CHEMOSENSITIVITY ASSAY 80
4.1.2.2 PREPARATION OF THE PGL4.29[LUC2P/CRE/HYGRO] VECTOR (MAXI-PREP)
AND
SEQUENCING 80
4.1.2.3 STABLE TRANSFECTION OF HEK293-SF-HH 4R-HIS6 CELLS WITH THE
PGL4.29[LUC2P/CRE/HYGRO] VECTOR 80
4.1.2.4 STABLE TRANSFECTION OF HEK293T CELLS WITH THE VECTOR
PGL4.29[LUC2P/CRE/HYGRO] 81
4.1.2.5 LUCIFERASE REPORTER GENE ASSAY IN THE 24-WELL FORMAT 81
4.1.2.6 LUCIFERASE REPORTER GENE ASSAY IN THE 96-WELL FORMAT 82
4.1.2.6.1 PREPARATION OF STOCK SOLUTIONS, DILUTION SERIES AND BUFFERS 82
4.1.2.6.2 PREPARATION OF THE CELLS 83
IMAGE 3
VII
4.1.2.6.3 DETERMINATION OF HH 4R LIGAND ACTIVITY 83
4.1.2.6.4 DETERMINATION OF NON-H 4R-MEDIATED LIGAND EFFECTS 84
4.1.2.6.5 DETERMINATION OF THE OPTIMAL FORSKOLIN CONCENTRATION 84
4.1.2.6.6 MONITORING THE TIME COURSE OF LUCIFERASE EXPRESSION 84
4.1.2.6.7 MEASUREMENT OF LUMINESCENCE WITH A MICROPLATE READER 84
4.1.2.7 AMINOPYRIMIDINES 85
4.1.3 RESULTS AND DISCUSSION 86
4.1.3.1 EFFECT OF HYGROMYCIN B ON HEK293-SF-HH 4R-HISB CELLS 86
4.1.3.2 TESTING AND SELECTION OF THE HEK293-SF-HH 4R -HIS6 -CRE-LUC
CELLS 87
4.1.3.3 ADAPTATION OF THE LUCIFERASE REPORTER GENE ASSAY TO THE 96-WELL
FORMAT 88
4.1.3.4 OPTIMIZATION OF THE PERIOD OF INCUBATION 89
4.1.3.5 OPTIMIZATION OF PRE-STIMULATION WITH FORSKOLIN 90
4.1.3.6 SELECTION OF THE HEK293-CRE-LUC CELLS 93
4.1.3.7 OFF-TARGET EFFECTS 94
4.1.3.8 FUNCTIONAL ACTIVITIES OF HISTAMINE H 4 RECEPTOR LIGANDS AT THE
HUMAN
HISTAMINE H 4R 95
4.2 DEVELOPMENT OF A REPORTER GENE ASSAY FOR THE MOUSE AND RAT HISTAMINE
H 4R 105
4.2.1 INTRODUCTION 105
4.2.2 MATERIAL AND METHODS 107
4.2.2.1 STABLE TRANSFECTION OF HEK293-SF-MH 4R-HIS6 CELLS WITH
PGL4.29[LUC2P/CRE/HYGRO] 107
4.2.2.2 PREPARATION OF THE PCDNA3.1(+)-SF-RH 4R-HIS6 VECTOR AND
SEQUENCING 107
4.2.2.3 STABLE TRANSFECTION OF HEK293-CRE-LUC CELLS WITH THE PCDNA3.1
(+)-SFRH 4R-HIS6 VECTOR 107
4.2.2.4 LUCIFERASE REPORTER GENE ASSAY 107
4.2.3 RESULTS AND DISCUSSION 108
4.2.3.1 SELECTION OF THE HEK293-SF-MH 4R-HISE-CRE-LUCTRANSFECTANTS 108
4.2.3.2 SELECTION OF THE HEK293-CRE-LUC-SF-RH 4R -HIS6 CELLS 109
4.2.3.3 INFLUENCE OF FORSKOLIN ON THE POTENCY OF HISTAMINE 110
4.2.3.4 FUNCTIONAL ACTIVITY OF HISTAMINE H 4 RECEPTOR LIGANDS AT THE
MOUSE AND RAT
HISTAMINE H 4 RECEPTOR 111
4.3 CONCLUDING REMARKS 124
4.4 REFERENCES 125
CHAPTER 5 DEVELOPMENT OF RADIOLIGAND BINDING ASSAYS FOR HUMAN AND RAT
HISTAMINE HI RECEPTORS 131
5.1 RADIOLIGAND BINDING ASSAY FOR THE HUMAN HISTAMINE H 3 RECEPTOR 132
IMAGE 4
132
133
133
134
134
134
140
140
141
141
142
142
143
.147
.148
.151
.152
.153
.153
.153
.153
.154
.154
.155
.165
.166
.169
INTRODUCTION
MATERIALS AND METHODS
WHOLE CELL RADIOLIGAND BINDING ASSAY
RESULTS AND DISCUSSION
SATURATION BINDING ASSAY
COMPETITION BINDING ASSAY
RADIOLIGAND BINDING ASSAY FOR THE RAT HISTAMINE H 3 RECEPTOR
INTRODUCTION
MATERIALS AND METHODS
WHOLE CELL RADIOLIGAND BINDING ASSAY
RESULTS AND DISCUSSION
SATURATION BINDING ASSAY
COMPETITION BINDING ASSAY
SUMMARY
REFERENCES
REPORTER GENE ASSAY FOR THE INVESTIGATION OF HUMAN AND RAT
HISTAMINE H 3 RECEPTOR LIGANDS
INTRODUCTION
MATERIAL AND METHODS
PREPARATION OF THE PCDNA3.1(+)-SF-RH 3R-HIS6 VECTOR AND SEQUENCING
STABLE TRANSFECTION OF HEK293-CRE-LUC WITH THE HUMAN AND RAT H 3R.
LUCIFERASE REPORTER GENE ASSAY
RESULTS AND DISCUSSION
SELECTION OF THE HEK293-CRE-LUC-SF-H/RH 3R-HIS6 CELLS
FUNCTIONAL CHARACTERIZATION OF LIGANDS AT THE HUMAN AND RAT HISTAMINE
H 3R
SUMMARY AND CONCLUSION
REFERENCES
SUMMARY
|
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| spelling | Nordemann, Uwe Verfasser aut Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs vorgelegt von Uwe Nordemann Radioligand binding and reporter gene assays for histamine H3 and H4 receptor species orthologs 2013 XI, 173 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2013 Histaminrezeptor (DE-588)4257968-5 gnd rswk-swf Ligand Biochemie (DE-588)4606532-5 gnd rswk-swf Isotopenmarkierung (DE-588)4176811-5 gnd rswk-swf Bindestelle (DE-588)4145563-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Histaminrezeptor (DE-588)4257968-5 s Ligand Biochemie (DE-588)4606532-5 s Isotopenmarkierung (DE-588)4176811-5 s Bindestelle (DE-588)4145563-0 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-280481 http://epub.uni-regensburg.de/28048/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-280481 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025931656&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Nordemann, Uwe Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs Histaminrezeptor (DE-588)4257968-5 gnd Ligand Biochemie (DE-588)4606532-5 gnd Isotopenmarkierung (DE-588)4176811-5 gnd Bindestelle (DE-588)4145563-0 gnd |
| subject_GND | (DE-588)4257968-5 (DE-588)4606532-5 (DE-588)4176811-5 (DE-588)4145563-0 (DE-588)4113937-9 |
| title | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs |
| title_alt | Radioligand binding and reporter gene assays for histamine H3 and H4 receptor species orthologs |
| title_auth | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs |
| title_exact_search | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs |
| title_full | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs vorgelegt von Uwe Nordemann |
| title_fullStr | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs vorgelegt von Uwe Nordemann |
| title_full_unstemmed | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs vorgelegt von Uwe Nordemann |
| title_short | Radioligand binding and reporter gene assays for histamine H 3 and H 4 receptor species orthologs |
| title_sort | radioligand binding and reporter gene assays for histamine h 3 and h 4 receptor species orthologs |
| topic | Histaminrezeptor (DE-588)4257968-5 gnd Ligand Biochemie (DE-588)4606532-5 gnd Isotopenmarkierung (DE-588)4176811-5 gnd Bindestelle (DE-588)4145563-0 gnd |
| topic_facet | Histaminrezeptor Ligand Biochemie Isotopenmarkierung Bindestelle Hochschulschrift |
| url | http://epub.uni-regensburg.de/28048/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-280481 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025931656&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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