| Project/Area Number |
07671279
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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 |
Embryonic/Neonatal medicine
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| Research Institution | Saitama Medical School |
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Principal Investigator |
SHIMIZU Hiroshi Saitama Medical School, Lecturer, 医学部, 講師 (90260843)
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| Co-Investigator(Kenkyū-buntansha) |
ARAKAWA Hiroshi Saitama Medical School, Instructor, 医学部, 助手 (90271238)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | pulmonary surfactant / small GTP binding protein / polymerase chain reaction / surfactant subtype conversion / surfactant convertase / surface area cycling / surfactant proteins / surfae area cycling / 新生児呼吸障害 / 肺胞II型細胞 / 低分子量GTP結合蛋白質 / 低分子量CTP結合蛋白質 |
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| Research Abstract |
Pulmonary surfactant is synthesized and secreted by alveolar type II epithelial cells. In this study we intended to (1) screen a rat lung cDNA library and isolate the small GTP binding proteins (rab proteins) which may be associated with surfactant trafficking in the type II cells, and (2) evaluate the surfactant subtype conversion as a metabolism of the surfactant.
A rat lung cDNA library was screened by a PCR-based method described by Amaravadi et al.[Primary screening] : Approximately 50000 plaque-forming units (pfu)/plate was plated on 150-mm plates. The phages were soaked from the plates. An aliquot from each phage stock was used for a PCR screening of the primary plates. A total of 1.9 x 10^6 cDNA clones (38 plates) were examined by PCR screening at a lower stringent condition using a previously isolated type II cell specific rab protein (smg24) primers, and nineteen positive plates were identified. Six of the nineteen positive plates were PCR positive at the lower stringent condi
… More
tion and negative at a higher stringent condition, suggesting these plates contain cDNAs homologous to the smg24. [Secondary screening] : One positive plate was chosen from the six primary plates for a secondary screening by replating at approximately 300 pfu/plate on 100-mm plates. Plates were treated as for the primary screening and PCR was performed. The PCR screening of the secondary plate lysates resulted in two positive plates. [Tertiary screening] : One positive aliquot was picked from the secondary plates, plated at a density of 100 pfu/plate and screened. The PCR screening of the tertiary plate identified six strong positive plates. The individual phage stocks were prepared from several single plaques and PCR was performed to isolate a single positive plaque. However, in spite of the strong positivity of the tertiary screening, the final PCR was unsuccessful to identify the positive stock. The reason is uncertain, however, this result may suggest the extremely slow growth rate of the phage which carries positive cDNA sequence. Further experiments such as the filter hybridization of the tertiary screening positive plates with the labeled PCR product are necessary to isolate positive clones.
In order to evaluate surfactant subtype conversion, we have developed in vitro surface area cycling technique. The surfactant subtype conversion was calculated as % conversion from the surface-active large aggregate surfactant to the surface-inactive small aggregate surfactant. Meconium increased the surfactant subtype conversion in a dose-dependent fashion, suggesting that meconium contains surfactant convertase-like activity. The surfactant convertase activity in meconium was blocked by a serine proteinase inhibitor, diisopropyl-fluorophosphate (DEP). We could also demonstrate that neutrophil elastase increases the surfactant subtype conversion in association with the degradation of surfactant protein A and B. Less
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