| Project/Area Number |
09044290
|
|---|
| Research Category |
Grant-in-Aid for international Scientific Research
|
| Allocation Type | Single-year Grants |
|---|
| Section | Joint Research |
|---|
| Research Field |
Pathological medical chemistry
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
TSUKITA Shoichiro Kyoto University Faculty of Medicine, Professor, 医学研究科, 教授 (50155347)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ITOH Masahiko Kyoto University Faculty of Medicine, Assistant, 医学研究科, 助手 (70270486)
TSUKITA Sachiko Kyoto University College of Medical Technology, Professor, 医療技術短期大学部, 教授 (00188517)
KUMAR Narlin Scrips Research Institute, Assistant, 助手
GILULA Norton Scrips Research Institute, Professsor, 主任教授
FURUSE Mikio Kyoto University Faculty of Medicine, Assistant, 医学研究科, 助手 (90281089)
ナーリン クマール スクリプス研究所, 助手
ノートン ギラール スクリプス研究所, 主任教授
ノートン ギルーラ スクリプス研究所, 主任教授
|
|---|
| Project Period (FY) |
1997 – 1998
|
| Project Status |
Completed (Fiscal Year 1998)
|
| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1998: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1997: ¥3,400,000 (Direct Cost: ¥3,400,000)
|
|---|
| Keywords | tight junction / occludin / claudin / cell adhesion / knockout / epithelial cells / intercellular junction / カドヘリン / アドヘレンスジャンクション / ES細胞 / 相同組換え / ドラッグデリバリー / 極性 / ターゲッティング |
|---|
| Research Abstract |
Occiudin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occiudin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from oceludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TI strands between wild-type and occludin-deficient epithelial cells. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures. We therefore re-examined the isolated junction fraction from chicken liver, from which occiudin was first identified. Among numerous components of this fraction,
… More
only a broad silver-stained band around 22 kD was detected with the occludin band through 4 M guanidine-HCI extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of data bases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 a.a., respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TI still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands. Less
|
