1997 Fiscal Year Final Research Report Summary
Investigation of cell biological function of a small G protein expressed in alveolar type II cells
| Project/Area Number |
08670685
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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 |
Respiratory organ internal medicine
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| Research Institution | Kanazawa Medical University |
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Principal Investigator |
OSANAI Kazuhiro Kanazawa Medical University, Department of Internal Medicine, Associate Professor, 医学部, 講師 (70221158)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Keiji Kanazawa Medical University, Department of Internal Medicine, Professor, 医学部, 教授 (50004685)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Small G protein / rab / alveolar type II cells / rat / localization |
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| Research Abstract |
Small GTP-binding proteins mediate a number of essential intracellular functions via participating' in cell signaling pathways. We investigated expression and localization of a novel rab protein in the lung tissue. cDNA of the protein had been cloned out from a rat lung cDNA library and recorded in Genbank by other researchers, but its function remains largely unknown. Recombinant protein was expressed in insect cells transfected with recombinant baculovirus carrying polyhistidine-tagged rab p24 cDNA, and was purified with a nickel affinity chromatography. Polyclonal antibody was raised against rabbits with a synthetic peptide designated from C-terminal amino acid sequence of the rab p24. Western blot using the antibody showed specific immunoreactive band corresponding to the predicted molecular weight of the rab p24 in total lung homogenate and isolated alveolar type II cells, but was not found in alveolar macrophage homogenate. Immunostaining of a frozen rat lung tissue revealed positive signal in airway epithelial cells and alveolar corner cells (possibly alveolar type II cells). Cell smear of isolated alveolar type II cells was also positive in immunostaining, whereas that of alveolar macrophages was negative. RT-PCR of total RNA extracted from purified alveolar type II cells was positive, whereas that of alveolar macrophages was negative, in situ hybridization using RNA riboprobe showed positive signals in alveolar corner cells and airway epithelial cells, especially in terminal airway cells (of which more than 80% are Clara cells in rats). Specific positive immunoreactive signal was seen in lysate of heavy vesicle fraction from isolated alveolar type II cells but was not seen in that of lamellar body fraction. These results showed that the rab p24 localizes in Clara cells and alveolar type II cells. Since these cells have distinct function, i.e. pulmonary surfactant metabolism, the protein may have a role in surfactant transport.
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