Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2003
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | München, Techn. Univ., Diss., 2003 |
| Beschreibung: | 115 S. Ill., graph. Darst. |
Internformat
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV017612914 | ||
| 003 | DE-604 | ||
| 007 | t | ||
| 008 | 031029s2003 ad|| m||| 00||| eng d | ||
| 016 | 7 | |a 969319878 |2 DE-101 | |
| 035 | |a (OCoLC)163193747 | ||
| 035 | |a (DE-599)BVBBV017612914 | ||
| 040 | |a DE-604 |b ger |e rakwb | ||
| 041 | 0 | |a eng | |
| 049 | |a DE-91 |a DE-12 | ||
| 100 | 1 | |a Puschmann, Andreas J. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |c Andreas J. Puschmann |
| 264 | 1 | |c 2003 | |
| 300 | |a 115 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a München, Techn. Univ., Diss., 2003 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010595359&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-010595359 | |
Datensatz im Suchindex
| _version_ | 1807682865061691392 |
|---|---|
| adam_text |
TABLE
OF
CONTENTS
LIST
OF
ABBREVIATIONS
.
6
1
BACKGROUND
.
7
1.1
CYTOKINE
SIGNALLING
.
7
1.2
HELICOBACTER
PYLORI
INFECTION
ACTIVATES
THE
HOST'S
IMMUNE
SYSTEM
IN
THE
GASTRIC
MUCOSA
.
8
1.3
TNF-A,
IL-1
P
OR
H
2
O
2
INFLUENCE
TRANSCRIPTION
IN
OTHER
TYPES
OF
CELLS
.8
1.4
NUCLEAR
FACTOR
KAPPA
B
(NF
K
B)
IS
A
WELL-CHARACTERISED
INDUCIBLE
TRANSCRIPTION
FACTOR
WHICH
IS
ACTIVATED
BY
INFLAMMATORY
STIMULI
.
10
1.4.1
NF
K
B:
MECHANISM
OF
ACTIVATION
.10
1.4.2
NF
K
B:
ACTIVATING
CONDITIONS
.
11
1.4.3
EFFECTS
OF
NF
K
B
ACTIVATION
.
12
1.4.4
NF
K
B
SUBUNITS
.
12
1.5
NF
K
B
IN
CELLS
OF
GASTRIC
ORIGIN
.
13
1.6
BACKGROUND:
SUMMARY
.
.
.
14
2
AIMS
.
15
3
METHODS
.
16
3.1
BACKGROUND
OF
METHODS
EMPLOYED
.
16
3.1.1
ISOLATION
OF
GASTRIC
EPITHELIAL
CELLS
.
16
3.1.2
ENRICHMENT
OF
PARIETAL
CELLS
.
17
3.1.3
VIABILITY
CONTROL
.
17
3.1.4
POLYMERASE
CHAIN
REACTION
FROM
REVERSELY
TRANSCRIBED
CDNA
(RT-PCR)
.
17
3.1.5
CELL
STIMULATION
.
18
3.1.6
CYTOSPINS,
CELL
CULTURE
AND
IMMUNOCYTOCHEMISTRY
.
18
3.1.7
PREPARATION
OF
NUCLEAR
EXTRACTS
AND
WHOLE
CELL
EXTRACTS
.
21
3.1.8
CONTROL
OF
NUCLEAR
EXTRACT
PURITY
.
22
3.1.9
ELECTROPHORETIC
MOBILITY
SHIFT
ASSAYS
(EMSAS)
.
23
3.2
EXPERIMENTAL
PART
.
26
3.2.1
ISOLATION
AND
ENRICHMENT
OF
PARIETAL
CELLS
.
26
3.2.2
RT-PCR
OF
MRNA
ISOLATED
FROM
PARIETAL
CELLS
.
27
3.2.3
IMMUNOCYTOCHEMISTRY
.28
3.2.3.1
IMMUNOSTAININGS
OF
FRESHLY
ISOLATED
PARIETAL
CELLS
WITH
THE
APAAP
DETECTION
METHOD
.
28
3.2.3.2
IMMUNOSTAININGS
OF
CULTURED
PARIETAL
CELLS
WITH
THE
FITC
DETECTION
METHOD
.
29
3.2.4
PREPARATION
OF
NUCLEAR
EXTRACTS
.30
3.2.5
DETERMINATION
OF
PROTEIN
CONCENTRATION
IN
NUCLEAR
EXTRACT
SAMPLES
.
32
3.2.6
DETERMINATION
OF
LACTATE
DEHYDROGENASE
(LDH)
ACTIVITY
.
32
3.2
7
ELECTROPHORETIC
MOBILITY
SHIFT
ASSAYS
(EMSAS)
.
35
3.2.7.1
RADIOLABELLING
OF
OLIGONUCLEOTIDES
.35
3.2.7
2
PREPARATION
OF
ELECTROPHORESIS
GELS
.
37
3.2.7.2.1
EXPERIMENTS
TO
QUANTIFY
NF
K
B
STIMULATION
.
38
3.2.7.2.2
COMPETITION
ASSAYS
.39
3
2
7.2.3
ANTIBODY
SUPERSHIFT
ASSAYS
.
41
3.2.7.2.4
MOLECULAR
WEIGHT
STANDARD
.
41
3.2.7.2.5
DRYING
OF
GELS
.
41
3.2.7.2.6
AUTORADIOGRAPHS
AND
EVALUATION
.42
3.2.8
TRANSFECTION
OF
CELLS
WITH
ANTISENSE
OLIGONUCLEOTIDES
.
43
13
MATERIALS
.
44
3.3.1
CHEMICALS
.
44
3.3
1.1
ISOLATION
OF
PARIETAL
CELLS
.
44
3.3.1.2
RT-PCR
.
44
3.3.1.3
CELL
STIMULATION
.
45
3.3.1.4
IMMUNOCYTOCHEMISTRY
.45
3.3.15
APAAP
IMMUNOSTAININGS
.
45
3.3.1.6
FITC
STAININGS
.
46
3.3.1.7
PREPARATION
OF
NUCLEAR
EXTRACTS
.
46
3.3.1
8
DETERMINATION
OF
PROTEIN
CONCENTRATION
.47
3.3.1.9
DETERMINATION
OF
LACTATE
DEHYDROGENASE
(LDH)
ACTIVITY
.47
3.3.1.10
ELECTROPHORETIC
MOBILITY
SHIFT
ASSAYS
(EMSAS)
.
47
3.3.1
11
AUTORADIOGRAPHSANDEVALUATION
.
49
3.3.1.12
TRANSFECTION
STUDIES
.49
3.3
2
BUFFER
SOLUTIONS
AND
MEDIA
.
50
3.3.2.1
ISOLATION
OF
PARIETAL
CELLS
.50
3.3.2.2
IMMUNOSTAININGS
OF
CULTURED
PARIETAL
CELLS
WITH
THE
FITC
DETECTION
METHOD
.
51
3.3.2
3
PREPARATION
OF
NUCLEAR
EXTRACTS
.
51
3.3.2.4
RADIOLABELLING
OF
OLIGONUCLEOTIDES
.52
3.3.2.5
PREPARATION
OF
ELECTROPHORESIS
GELS
.52
3.3.2.6
PROTEIN/DNA
BINDING
REACTIONS
.53
4
RESULTS
.
54
4.1
RT-PCR
.54
4.2
IMMUNOCYTOCHEMICAL
STUDIES
.54
4.2.1
IMMUNOCYTOCHEMICAL
STAININGS
OF
FRESHLY
ISOLATED
PARIETAL
CELLS
WITH
THE
APAAP
DETECTION
METHOD
.54
4.2.2
IMMUNOSTAININGS
OF
CULTURED
PARIETAL
CELLS
WITH
THE
FITC
DETECTION
METHOD
.57
3
4.3
ELECTROPHORETIC
MOBILITY
SHIFT
ASSAYS
(EMSAS)
.
62
4.3
1
COMPETITION
ASSAYS
.
62
4.3.2
QUANTITATIVE
EMSA
STUDIES
.63
4.3.3
ANTIBODY
SUPERSHIFTS
.
71
4.3.4
INHIBITION
OF
NF
K
B
P65
EXPRESSION
BY
ANTISENSE
PHOSPHOROTHIOATE
OLIGONUCLEOTIDES
DIRECTED
AGAINST
THE
P65
SUBUNIT
GENE
.
72
5
DISCUSSION
.
75
5.1
BACKGROUND
OF
STUDY
DESIGN
.75
5.1.1
MONOCYTES/MACROPHAGES
PRODUCE
THE
PRO-INFLAMMATORY
CYTOKINES
TNF-A
AND
IL-1
P
.
76
5.1.2
NEUTROPHILS
RELEASE
REACTIVE
OXYGEN
INTERMEDIATES
.
77
5.1.3
ELEVATED
MUCOSAL
LEVELS
OF
TNF-A
ARE
ALSO
DETECTED
IN
NSAID-INDUCED
FORMS
OF
GASTRITIS
.78
5.2
DISCUSSION
OF
METHODS
.78
5.2.1
USE
OF
ISOLATED
RAT
PARIETAL
CELLS
.
78
5.2.2
PREPARATION
OF
HIGH-PURITY
NUCLEAR
EXTRACTS
.78
5.2.2.1
METHOD
AND
CONTROLS
.
79
5
2.2.2
COMPARISON
TO
METHODS
OF
CELL
EXTRACT
PREPARATION
DESCRIBED
BY
OTHERS
.79
5.2.2.3
EMSAS
AND
QUANTITATIVE
EVALUATION
.
80
5.2.2.4
SP1
AS
STANDARD
FOR
QUANTITATION
.80
5.3
DISCUSSION
OF
RESULTS
.
81
5.3.1
SUBTYPE
1
TNF-A
RECEPTOR
(CD
120A;
TNFR1)
IS
EXPRESSED
BY
PARIETAL
CELLS
.
81
5.3.2
NF
K
B
IS
PRESENT
IN
ISOLATED
PARIETAL
CELLS
.82
5.3.3
NF
K
B
IN
ISOLATED
PARIETAL
CELLS
IS
ACTIVATED
BY
REACTIVE
OXYGEN
INTERMEDIATES
(H2O2)
OR
THE
INFLAMMATORY
CYTOKINES
IL-1
P
OR
TNF-A
.
83
5
3.3.1
EVIDENCE
FROM
IMMUNOCYTOCHEMICAL
EXPERIMENTS
IN
THE
PRESENT
STUDY
.83
5.3.3.2
EVIDENCE
FROM
EMSAS
IN
THE
PRESENT
STUDY
.84
5.3.3.3
PREVIOUS
STUDIES
INVESTIGATING
NF
K
B
ACTIVATION
BY
TNF-A,
IL-1
P
OR
REACTIVE
OXYGEN
INTERMEDIATES
IN
OTHER
CELL
SYSTEMS
.
84
5.3.3.4
OTHER
STUDIES
INVESTIGATING
NF
K
B
ACTIVATION
IN
GASTRIC
EPITHELIAL
CELLS
ACTIVATED
BY
HELICOBACTER
PYLORI
.86
5.3.3.5
PREVIOUS
STUDIES
INVESTIGATING
NF
K
B
ACTIVATION
IN
GASTRIC
EPITHELIAL
CELLS
BY
VARIOUS
OTHER
SUBSTANCES
.
86
5.3.3.6
CONCLUSION
.
86
.
5
3.4
THE
ACTIVATION
OF
NF
K
B
BY
THESE
STIMULI
WAS
ASSESSED
QUANTITATIVELY
.87
5.3.4.1
RESULTS
FROM
QUANTITATIVE
EXPERIMENTS
IN
THE
PRESENT
STUDY
.
87
4
5.3.4.2
PREVIOUS
STUDIES
ON
TIME
AND
CONCENTRATION-DEPENDENT
ACTIVATION
BY
TNF
A,
IL-1
P,
OR
H
2
O
2
IN
CELLS
OF
GASTRIC
ORIGIN
.
87
5.3.4.3
PREVIOUS
STUDIES
DETERMINING
THE
CONCENTRATION
OF
TNF-A,
IL-1
P,
OR
REACTIVE
OXYGEN
INTERMEDIATES
IN
INFLAMED
GASTRIC
MUCOSA
.88
5
3.4.4
PREVIOUS
STUDIES
QUANTIFYING
NF
K
B
ACTIVATION
.88
5.3.4.5
CONCLUSION
.89
5.3.5
THE
SUBUNIT
COMPOSITION
OF
THE
NF
K
B
DIMERS
INVOLVED
HAS
BEEN
DETERMINED
.
90
5.3.5.1
NF
K
B
SUBUNITS
IN
MAMMALIAN
CELLS
.
90
5.3.5.2
FUNCTIONAL
DIFFERENCES
OF
THE
NF
K
B
SUBUNITS
.
91
5.
3.5.2.1
DIFFERENT
AFFINITY
TO
DNA
SITES
.
91
53.5.2.2
DIFFERENT
POTENCY
TO
ACTIVATE
THE
RATE
OF
TRANSCRIPTION
.
91
5.3.5.3
SUBUNIT-SPECIFIC
INTERACTION
OF
NF
K
B
WITH
DIFFERENT
I
K
B
ISOFORMS
.
91
5.3.5.4
SUBUNIT
ANALYSIS
OF
NF
K
B
DIMERS
IN
THE
PRESENT
STUDY
.
92
53.5.4.
1
P65
AND
P50
SUBUNITS
IN
THE
PRESENT
STUDY
.93
53.5.4.2
P65
AND
P50
IN
PREVIOUS
STUDIES
WITH
GASTRIC
EPITHELIAL
CELLS
.93
53.5.4.3
P52
SUBUNIT
IN
THE
PRESENT
STUDY
.
94
53.5.4.4
PREVIOUS
STUDIES
ON
P52
IN
OTHER
CELL
TYPES'
.96
53.5.4.5
P52
IN
GASTRIC
EPITHELIUM
IN
PREVIOUS
STUDIES
.96
53.5.5
NF
K
B
SUBUNITS:
CONCLUSION
.98
5.3.6
TRANSFECTION
OF
ISOLATED
RAT
PARIETAL
CELLS
WITH
ANTISENSE
OLIGONUCLEOTIDES
AGAINST
THE
NF
K
B
P65
SUBUNIT
GENE
EFFICIENTLY
SUPPRESSES
P65
EXPRESSION
.
98
6
CONCLUSION
.
100
7
REFERENCES
.
102
5 |
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| author | Puschmann, Andreas J. |
| author_facet | Puschmann, Andreas J. |
| author_role | aut |
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| building | Verbundindex |
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| ctrlnum | (OCoLC)163193747 (DE-599)BVBBV017612914 |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV017612914 |
| illustrated | Illustrated |
| indexdate | 2024-08-18T00:25:18Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-010595359 |
| oclc_num | 163193747 |
| open_access_boolean | |
| owner | DE-91 DE-BY-TUM DE-12 |
| owner_facet | DE-91 DE-BY-TUM DE-12 |
| physical | 115 S. Ill., graph. Darst. |
| publishDate | 2003 |
| publishDateSearch | 2003 |
| publishDateSort | 2003 |
| record_format | marc |
| spelling | Puschmann, Andreas J. Verfasser aut Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells Andreas J. Puschmann 2003 115 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2003 (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=010595359&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Puschmann, Andreas J. Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |
| subject_GND | (DE-588)4113937-9 |
| title | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |
| title_auth | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |
| title_exact_search | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |
| title_full | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells Andreas J. Puschmann |
| title_fullStr | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells Andreas J. Puschmann |
| title_full_unstemmed | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells Andreas J. Puschmann |
| title_short | Characterisation of the functional role of the inducible transcription factor nuclear factor kappa-B (NF-kappaB) in isolated rat parietal cells |
| title_sort | characterisation of the functional role of the inducible transcription factor nuclear factor kappa b nf kappab in isolated rat parietal cells |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010595359&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT puschmannandreasj characterisationofthefunctionalroleoftheinducibletranscriptionfactornuclearfactorkappabnfkappabinisolatedratparietalcells |