| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
|
|---|
| Research Abstract |
We designed a new medium (YIGADHA-S) based on the casin-free YI-S medium (Diamond, 1995) and the monoxenic culture system with Pseudomonas aeruginosa (Kobayashi et al, 1998) for trail of axenic cultivation of Entamoeba dispar YIGADRA-S is deferent from YI-S in that glucose is replaced by gluconic acid and dihydroxyactone, and the medium is sterilized by filtration. Consequently this YIGADHA-S made successful axenic culture of four E. dispar strains, However the growth of E. dispar strains were very poor except for one strain, We recently found that various cell lines from lots of species (over 18 species) of animal or plant origin, which were autoclaved (121℃, 15min.), also could support the growth of E. dispar in addition to bacteria (Eschericia coli, P. aeruginosa and others) and Crithidia fasciculate, and found an interesting property common to every cell (e.g. micro organisms, protozoan, mammalian and plant cells) for supporting the growth of E. dispar in YIGADHA-S, That is, those
… More
cells except for bacteria had mitochondria, hydrogenosome or mitochondria and chroloplast. On the other hand, some protozoan (Entamoeba histolytica, E. dispar, E. moshkovskii (Laredo), E. invadens (IP-1), Ciardia lamblia (Portland I) cells and human erythrocyte which lacked mitochondria or mitochondria like organelles didn't make support the growth of E. dispar in this medium. We supposed from these results that the common nonheme iron-sulfur protein existing in mitochondria, hydrogenosome, chroloplast and bacteria might support the growth of E. dispar. And the effect of purified ferredoxin (Sigma) from spinach (ferredoxin is known as an essential redox protein in the energy metablism of E. histolytica) on the growth of E. dispar was tested. It was confirmed that ferredoxin (2.5 μg/ml【less than or equal】) had a favorable effect on the growth of E. dispar. And we developed a simple method for extraction of ferredoxin-rich fraction from isolated and washed intact leaf cells containing many chroloplasts of spiderwort (Commelina communis L.) by sonication. Consequently, this ferredoxin-rich extract made successful axenic culture of five E. dispar strains at similar maximum growth rate (constantly 3-4x10ィイD14ィエD1/ml). And' we also established an in vivo system for infection of E. dispar in caecum by using Rag gene knockout immunodeficient mouse (BALB/c Rag -/-), though the period of infection was for 3 days. Less
|
