| Project/Area Number |
17591105
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Pediatrics
|
|---|
| Research Institution | NAGOYA CITY UNIVERSITY |
|---|
Principal Investigator |
ASAI Kiyofumi Nagoya City University, Graduate School of Medical Sciences, Associate Professor, 大学院医学研究科, 教授 (70212462)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MIURA Yutaka Nagoya City University, Graduate School of Medical Sciences, Associate Professor, 大学院医学研究科, 助教授 (90285198)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Keywords | Glia / Astrocyte / GMFB / GMFG / Microarray |
|---|
| Research Abstract |
1, Analysis of expression of glia maturation factor beta after cryogenic injury
We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury.
2, Analysis of promoter activity of the human glia maturation factor-gamma gene
We determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region-84 to-70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells.
We have also determined the organization of the mouse GMFG gene and cloned the 5'-flanking region of the first exon. The promoter activity was analyzed using luciferase assay.
|
