| Budget Amount *help |
¥15,500,000 (Direct Cost: ¥15,500,000)
Fiscal Year 2004: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2003: ¥10,500,000 (Direct Cost: ¥10,500,000)
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| Research Abstract |
(1) We isolated a cDNA clone encoding znCD by 5' and 3' RACE-PCR. It possessed an open reading fame of 2,229 base pairs encoding 743 amino acids. The enzyme activity at neutral pH markedly increased after transformation of Chinese hamster CHOP and zebrafish BRF41 cells with the cDNA. The overexpressed enzyme was found to be distributed in ER/Golgi compartments as well as the plasma membranes. Antisense morpholino oligonucleotide (AMO), which was designed based on the sequence of znCD mRNA, successfully blocked the translation of znCD in a wheat germ in vitro translation system. The knockdown of znCD with AMO led to an increase in the number of zebrafish embryos with severe morphological and cellular abnormalities ; abnormal morphogenesis in the head and tail, pericardiac edema, defect of blood cell circulation, and increase of apoptotic cells especially in the head and neural tube regions at 36 h postfertilization. The ceramide level in AMO-injected embryos significantly increased comp
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ared to that in control embryos. Simultaneous injection of both AMO and synthetic znCD mRNA into 1 cell-stage embryos rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and early development of zebrafish.
(2) Endoglycoceramidase (EGCase ; EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. The hydra EGCase, consisting of 517 amino acid residues, showed 19.2% and 50.2% identity to the Rhodococcus and jellyfish EGCases, respectively. The recombinant hydra enzyme, expressed in CHOP cells, hydrolyzed [^<14>C]GM1a to produce [^<14>C]ceramide with a pH optimum at 3.0-3.5. Whole mount in situ hybridization and immunocytochemical analysis revealed that EGCase was widely expressed in the endodermal layer, especially in digestive cells. GM1a injected into the gastric cavity was incorporated and then directly catabolized by EGCase to produce GM1a-oligosaccharide and ceramide, which were further degraded by exoglycosidases and ceramidase, respectively. However, hydra exoglycosidases did not hydrolyze GM1a directly. These results indicate that the EGCase is indispensable for the catabolic processing of dietary glycosphingolipids in hydra, demonstrating the unique catabolic pathway for GSLs in the animal. Less
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