|Budget Amount *help
¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 1999 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1998 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Cytokines were originally reported to be generated during the interaction of lymphocytes with specific antigens or non-specific mitogens and to function as communication signals among the immune systems. To investigate an interaction between lymphocytes and neurons or endocrine cells, and to seek for a new cytokine, the effects of lymphocytes and their conditioned medium on catecholamine efflux were examined in cultured bovine adrenal medullary cells. 1) Co-culture of adrenal medullary cells with lymphocytes for 3 days enhanced catecholamine efflux from the cells. 2) A conditioned medium prepared by incubation of lymphocytes for 3 days also stimulated catecholamine efflux in a time-and concentration-dependent manner. 3) Heat treatment of the conditioned medium at 60℃ and 100℃ reduced stimulatory effect to 59 and 20% of control. The stimulatory activity of the conditioned medium was almost observed in a high molecular fraction after dialysis (<8kDa cutoff) or gel filtration. 4) The cond
itioned medium had little effect on the activity of lactate dehydrogenase in the medium of cultured adrenal medullary cells and on desipramine-sensitive [ィイD13ィエD1H] norepinephrine uptake by the cells. These findings suggest that lymphocytes release a heat-sensitive and high molecular (>8kDa) factor (lymphocyte-derived factor) which stimulates catecholamine efflux from adrenal medullary cells.
Lymphocyte-derived factor(s)(LDFs) were partially purified by precipitation with 100% ammonium sulfate and followed by DEAE-cellulose column chromatography. Three peaks of active fraction were collected and pooled. Two antisera against the peak l and peak 2 were prepared and named as fuzzy antibody land antibody 2. Using the fuzzy antibody 1, 20 positive clones were isolated from bovine spleen cDNA library and partially sequenced. In these clones sequenced, the sequences of several clones (NY105, NY10, NY11) were new one when searched by the GENBANK or EMBL databases. The clones of NY16, NY18 and NY5 were found to be a new isoform of IgM, intron region for ornithine decarboxylase and one kind of serine protease, respectively.
リンパ球由来因子を生化学的に部分精製し、その活性フラクションに対する抗体(ファジー抗体)をウサギにて作成した。リンパ球由来因子を生化学的手法(Sephacryl S-300カラムクロマトグラフィー、高速液体クロマトグラフィー等)で精製を試みたが、完全に精製するには至らなかった。そこで、上記のファジー抗体を用いて、ウシ脾臓cDNAライブラリィーから20ヶの陽性反応を示すクローンを分取した。その各々のクローンの部分塩基配列を解読して既知蛋白質とのホモロジー検索を行ったところ、全く報告されていない塩基配列をもった3ヶのクローン(NY10,NY11及びNY105)が見つかった。また、3ヶのクローン(NY16,NY18及びNY5)のさらに長い塩基配列を解読した結果、IgMの新しいisoform, ornithine decarboxylaseのあるイントロン領域、そしてserine proteaseの一種であることが判明した。