コプラナーPCBsによるABCタンパク質機能阻害がxenobioticsの細胞毒性およびステロイドホルモン代謝に与える影響

イヌ扁平上皮がん由来細胞およびイヌ線維肉腫由来細胞からcolchicine耐性株として選択した細胞へのPgp発現をwestern blotおよび定量的PCRで確認した。各薬剤耐性株へのPgp発現が,タンパク質およびmRNAレベルで確認され,Co-PCBsのPgp影響モデルの1つが確立された。Pgp発現細胞の膜標品をN_2ガス破砕法で調製し,そのATPase活性をATP regenerating系で測定した。各阻害剤および活性化剤を用いPgp-ATPaseほかNa,K-ATPaseおよびCa-ATPase活性測定が可能であった。しかし,Pgp-ATPase活性は極めて低く,本測定法ではCo-PCBs阻害の速度論的解析は困難であると判断した。したがって,次年度はPgpを精製し,そのPgp-ATPase活性を測定することを試みることにした。Expression of Pgp was confirmed by western blot and quantitative PCR in canine squamous cell carcinoma and canine fibrosarcoma which were selected as colchicine tolerance cell line. The protein and mRNA of Pgp were found in the cells, thus one of the examination models for the effect of Co-PCBs on Pgp was established. The membrane was prepared by N_2 disruption from Pgp expressed cells, and the ATPase was measured by ATP regeneration system. The activities of Pgp-ATPase, Na,K-ATPase and Ca-ATPase were measured by adding with the inhibitor and/or the activator. However, Pgp-ATPase activity was too small to analyze kinetics of the inhibition with Co-PCBs. Therefore, we are going to purify Pgp and measure its ATPase activity in the next year

精神疾患関連遺伝子多型の解析

ヒトGSK3β遺伝子のプロモーター領域について多型解析を行った結果,統合失調症および躁鬱病と有意に相関する遺伝子多型は認められなかった。このことは,mRNAの安定性に関与する領域,トランス調節因子などについて解析する必要があることを示唆している。The aim of this study is to reveal polymorphism in genes associated with psychiatric disorders and neurodegenerative diseases. Genomic DNA from patients affected by schizophrenia or bipolar, and normal humans were analyzed. Comparing the psychiatric disorders with normal humans, no significant differences in a ratio of polymorphism found throughout the regulatory region of GSK3β gene (promoter and 5\u27-UTR). Lithium, a therapeutic agent for psychiatric disorder such as bipolar, inhibits GSK3βand production of amyloidβ, a main component of senile plaques in Alzheimer\u27s disease. In addition, the level of GSK3βis decreased in schizophrenia patients and increased depending on aging. The results in this study lead us to consider that 3\u27-UTR of GSK3β gene and trans acting factors for GSK3β transcription should be studied. In conclusion, the present data may provide us basic and important information that promote revealing the molecular mechanism for regulation of GSK3βgene expression