| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Helicobacter pyroli is a gram-negative microaerobic bacterium, which has been proposed to be a cause of peptic ulcers. Laboratory growth of H.pyroli is carried out by using complex media containing serum, and blood derivatives. In stead, H.pyroli can be grown in media containing β-cyclodextrin. The reason of this fact is unknown yet, but it is suggested that CD traps the substances that is harmful for H.pyroli growth. If the trapped material can be identified, it will be a good lead compound for the development of the inhibitor of H.pyroli growth. From this point of view, we tried to isolate the CD-guest complex from the supernatant of the media used for the culture of H-pyroli. However, CD-guest complex had no UV absorption, causing its purification extremely difficult.
In this research, we synthesized three β-CD derivatives, which will facilitate the purification of CD-guest complex. First compound is biotinylated β-CD. This CD is synthesized to purify the complex by avidin column. Ho
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wever, this compound was highly insoluble in water and thus, could not be used for the culture of H.pyroli. Next, we prepared β-CD carrying 4-bromo-2, 3, 5, 6-tetrafluorobenzoyl group. Bromine is composed of two isotopes, ^<79>Br and ^<81>Br with almost the same amount. Thus, elution fraction of this CD derivative will be easily identified by the characteristic mass spectrum derived from bromine. Although the culture of the H.pyroli using this CD was successful, the elution position of this CD derivative could not be identified by the mass spectrum. The last derivative, which carries fluorescein on β-CD, was also successfully used for the culture of H.pyroli. The post-cultural supernatant was separated by gel-filtration chromatography. The elution position of the CD derivative was clearly identified by its strong fluorescence and UV absorbance. The analysis of fraction containing the CD derivative by MALDI mass showed that the signal, which is about 100 atomic mass unit higher than the CD derivative was observed. This result might show that the CD derivative traps a substance, which has a molecular weight of about 100. We are now trying to identify its structure by ^1H-NMR.
In conclusion, we succeeded in establishing a method to facilitate the purification of CD-guest compound from the cultural supernatant of H.pyroli, which will be useful to identify the growth inhibitor of H.pyroli. This method will be also applicable to find the inhibitor of other bacteria, such as B.pertussis, which can also be cultured in β-CD-containing media. Less
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