1997 Fiscal Year Final Research Report Summary
Mechanisms of Bacterial Endotoxin-induced Cell Activation by a Serum-independent Pathway.
| Project/Area Number |
08670315
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Jichi Medical School |
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Principal Investigator |
KIRIKAE Teruo Jichi Medical School, Department of Microbiology, Assistant Professor, 医学部, 講師 (50192563)
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| Project Period (FY) |
1996 – 1997
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| Keywords | ENDOTOXIN / LPS / CD14 / ST2 CELLS / IL-6 / モノクロナール抗体 |
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| Research Abstract |
The present study was done to elucidate the biological properties of LPS receptor (s) and its signal transduction system, and to identify some molecules of them. A cell line, ST2, derived from murine bone marrow stroma, was mainly used in the present study. We examined the following points ; a) responsiveness to LPS antagonists and LPS agonists, b) detection of LPS-binding proteins on the ST2 cells and production of polyclonal antisera against these proteins, c) detection of tyrosine-phosphorylated proteins on the ST2 cells. We identified some molecules which is related to LPS-induced activation of ST2 cells.
1) Adaptation of CD14-negative marrow stromal ST2 cells in serum-free medium and LPS-responsiveness of ST2 cells
To exclude completely the effects of CD14 and serum components on LPS responsiveness, we established a subclone of CD14-negative marrow stromal ST2 cells growing under serum-free conditions.
2) Detection of LPS-binding proteins on ST2 sells growing under serum-free conditions.
A ligand-botting method and a cross-linker method using radiolabelled LPS demonstrated a number of LPS-binding proteins on ST2 cells.
3) Production of polyclonal antisera against membrane fraction containing LPS-binding proteins on ST2 cells.
Rabbits were immunized with partial purified LPS-binding proteins on ST2 cells and polyclonal antisera were collected. The biological immunological properties were determined.
4) Detection of tyrosine-phosphorylated proteins on ST2 sells growing under serum-free conditions.
A immunoprecipitation and western blotting methods revealed tyrosine-phosphorylated proteins on ST2 sells stimulated with LPS.
5) Production of monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.
On based our previous finding, we produced monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.
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