| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
The aim of this study was a detection of a murine granulysin. cDNA library was initially constructed using mRNA derived from small intestine cells and spleen cells of mice. Subsequently, a hybridization with a probe of human granulysin gene was performed to detect a murine counterpart. Several positive clones from the cDNA library were obtained after the hybridization followed by gene sequence analysis of the clones. However, all of the clones failed to show a similar sequence to that of human granulysin. Then, a cloning of murine granulysin was carried out by RT-PCR using primers encompassing a 9kDa of human granulysin as well as primers from porcine granulysin. As a template, total RNAs derived from cells of various organs of mice were used. PCR products obtained by the RT-PCR were examined for the sequences. Nonetheless, a mouse homologue of granulysin was unable to be detected. Furthermore, a southern hybridization was performed with a genomic cDNA as a template of mice to detect a
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promoter region of a murine granulysin. A promoter sequence of human granulysin was employed as a probe. However, no cDNA clone similar to the human granulysin was obtained, either. Thus, a murine granulysin was to date not obtained yet. We assume that the level of granulysin expression in mice was highly low, which may be a reason for failure of cloning a murine granulysin. It is noteworthy that there's at present no reports concerning on the murine granulysin.
In addition to the above experiments, we examined the regulatory mechanisms underlying human granulysin gene expression using a human monocytic cell line THP-1 treated with Acholeplasma laidlawil We found that AP-1 and C/EBPβ, but not NF-kB participate in the regulation of inducible granulysin gene expression in THP-1 cells. Interestingly, the gene expression of granulysin was found to be exquisitely regulated by positive (AP-1) and negative (C/EBPβ) transcription factors. As for functional analysis of granulysin, we synthesized a small linear cationic peptide (residues 32-42) derived from human granulysin. The small cationic peptide displayed an antimicrobial activity against gram positive and negative bacteria, including S. aureus and E. coll. Less
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