Molecular cloning and characterization of a novel sequences with enhanced expression in permanent ischemic rat orain

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To clarify the molecular mechanisms of generation of dementia caused by chronic cerebral hypoperfusion, some kinds of intrinsic factors which changed the mRNA expression were cloned by differential display from the rat brain treated by permanent occlusion of bilateral common carotide arteries (2VO). Among them, further detail studies were done for two novel factors which expressions were enhanced by 2VO. One was isolated as a factor that the expression was enhanced at 4 days after 2VO, and named vof-21 (accession No. AB089205). Whole sequence of vof-21, 4,885 nt, was clarified. The eatimated amino acid sequence was 265 residues. The expression was shown a peak at 7 days after 2VO, and thendecreased to the sham level at 35 days. The expression was abundant in brain and lung followed by in thymus. The results of in situ hybridization revealed that the expressions in brain were rich in locus coreuleus, pontine nucleus and hippocampus. Another one was enhanced the expression at 4 months af … More ter 2VO and called vof-16 (accession No. AB089206). The whole sequence was 2,098 nt and the eatimated amino acid sequence was 73 residues. The vof-16 was abundant in the hippocampus, the tenia tecta, the piriform cortex and the area around the aorta. The expression was abundant in the hippocampus of rats with cognitive impairment induced by 2VO. In contrast, the expression levels of vof-16 were lower in the 2VO rats with no impairment and in sham-operated rats. The RT/PCR show that both vof-16 and vof-21 mRNA also existed in NG108-15 cells. To inhibit the expression by delivering the antisense DNA into the cell, the conditions of pH-sensitive liposome methods were established and reported. The functional sygnificance of vof-21 was still studying by using of this liposome system especially on Ca^<2+> and other ion channel and/or mobility. New methods for these kinds of experiment were also developed and reported ; the improved methods of 5'-RASE and determination methods of sense sequence based on RT/PCR. Involvement of 5-HT2C receptor and the variant on neuronal differentiation of NG108-15 cells was also studied by this grant. Less