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9. The involvement of Cry1 and Cry2 genes in the regulation of the circadian body temperature rhythm in mice

Author

Nagashima, Kei, Matsue, Kenta, Konishi, Masahiro, Iidaka, Chisato, Miyazaki, Koyomi, Ishida, Norio, and Kanosue, Kazuyuki

Subjects
Mice as laboratory animals -- Research, Circadian rhythms -- Effect of chemicals on, Metabolism, Body temperature, Biological rhythms, Biological sciences
Abstract

The criptochrome genes (Cry1 and Cry2) are involved in the molecular mechanism that controls the circadian clock, and mice lacking these genes ([Cry1.sup.-/-]/ [Cry2.sup.-/-]) are behaviorally arrhythmic. It has been speculated that the circadian clock modulates the characteristics of thermoregulation, resulting in body temperature ([T.sub.b]) rhythm. However, there is no direct evidence proving this speculation. We show here that Tb and heat production in [Cry1.sup.-/-]/[Cry2.sup.-/-] mice are arrhythmic under constant darkness. In contrast, both rhythms occur under a light-dark cycle and/or periodical food restriction linked with spontaneous activity and/or eating, although they are not robust as those in wild-type mice. The relationship between heat production and [T.sub.b] in [Cry1.sup.-/-]/[Cry2.sup.-/-] mice is linear and identical under any conditions, indicating that their [T.sub.b] rhythm is determined by heat production rhythm associated with activity and eating. However, [T.sub.b] in wild-type mice is maintained at a relatively higher level in the active phase than the inactive phase regardless of the heat production level. These results indicate that the thermoregulatory responses are modulated according to the circadian phase, and the Cry genes are involved in this mechanism. circadian clock; thermoregulation; metabolism

Published
2005

17. Novel and Stable Dual-Color IL-6 and IL-10 Reporters Derived from RAW 264.7 for Anti-Inflammation Screening of Natural Products

Author

Saiki, Papawee, Kawano, Yasuhiro, Nakajima, Yoshihiro, Van Griensven, Leo J.L.D., Miyazaki, Koyomi, Saiki, Papawee, Kawano, Yasuhiro, Nakajima, Yoshihiro, Van Griensven, Leo J.L.D., and Miyazaki, Koyomi

Abstract

Our previous study suggested that the interleukin (IL)-6 and IL-10 could serve as good biomarkers for chronic inflammatory disease. We previously established an IL-6 and IL-10 reporters assay that could examine reporter activity along with the reference gene in LPS-induced RAW 264.7 cells. In this study, we described new and stable RAW 264.7 derived dual-color IL-6/gapdh and IL-10/gapdh reporters. This assay allowed us to easily determine relative IL-6 and IL-10 levels with 96-well plate within one step. We evaluated the relative IL-6 and IL-10 levels in the LPS-induced stable cells testing 52 natural products by real-time bioluminescence monitoring and time-point determination using a microplate luminometer. The relative IL-6 and IL-6/IL-10 values decreased by the crude ethanol extracts from nutmeg and by 1'S-1'-acetoxychavicol from greater galangal using real-time bioluminescence monitoring. At the same time, the relative IL-10 was induced. The relative IL-6 and IL-6/IL-10 decreased by crude ethanol extracts from nutmeg and 1'S-1'-acetoxychavicol acetate at 6 h. Only crude ethanol extract from nutmeg induced IL-10 at 6 h. We suggested that the use of these stable cells by real-time monitoring could serve as a screening assay for anti-inflammatory activity and may be used to discover new drugs against chronic inflammatory disease.

Published
2019

32. Effect of lipopolysaccharide on circadian clock genes Per2and Bmal1in mouse ovary

Author

Shimizu, Takashi, Watanabe, Kaya, Anayama, Nozomi, and Miyazaki, Koyomi

Abstract

In mammals, circadian rhythms are associated with multiple physiological events. The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on circadian systems in the ovary. Immature female mice were received an intra-peritoneal injection of equine chorionic gonadotropin (eCG) and LPS. Total RNA was collected from the ovary at 6-h intervals throughout a 48 h of experimental period. The expression of the circadian genes period 2(Per2) and brain and muscle ARNT-like 1(Bmal1) such as circadian genes was measured by quantitative PCR. Although expression of Per2and Bmal1in the ovary did not display clear diurnal oscillation, LPS suppressed the amplitude of Per2expression. Additionally, LPS inhibited the expression of cytochrome P450 aromatase(CYP19) and luteinizing hormone receptor(LHr) genes in the ovary of eCG-treated mice. Our data suggest that Per2may be associated with the inhibition of CYP19and LHrexpression by LPS in the ovaries of immature mice.

Published
2017
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37. 細胞接着性タンパク質ビトロネクチンの構造と機能に関する研究

Author

Miyazaki, Koyomi

Abstract

text/plain, application/pdf, 学位論文, ピトロネクチンは血漿、血小板、細胞外マトリックスなどに存在する糖タンパク質で、動物細胞を基質に接着・伸展させる活性の他、血液凝固・線溶系や免疫補体系での調節機能を合わせ持っている。今まで、ヒトのピトロネクチンについてはその構造と機能の研究が盛んに行われてきたが、その他の生物のピトロネクチンについてほとんど研究されていなかった。そこで、様々な生物のピトロネクチンの構造と機能を研究し、生物界全体に普遍的なピトロネクチンの統一的な構造と機能を明らかにすることにした。\\r\\\ まず、様々な生物にピトロネクチンが存在しているかどうかを調べた。その1つの方法として、私の研究室で開発されたピトロネクチンの簡便精製法に従がい、14種の哺乳類、鳥類などの高等動物血漿よりピトロネクチンを精製した。精製されたピトロネクチンは、分子量が58－78 kDaで、バンドの数も1－3本と多様性がみられた。また、もう1つの方法として、ヒト、ウシ、ブタ、ニワトリのピトロネクチンに対する抗体を調製し、その他の生物におけるピトロネクチン様タンパク質の存在を抗体反応性で検討した。すると、両生類、ハ虫類、魚類などの脊椎動物、ショウジョウバエや線虫などの無脊椎動物、真性粘菌といった粘菌類にまでピトロネクチン様タンパク質が見いだされた。ピトロネクチン様タンパク質の分子量は、51－96 kDaで、バンドの数も1－3本と精製動物ピトロネクチンと同様に多様であった。\\r\\\ ピトロネクチンの統一分子像を考えるためには、精製動物ピトロネクチンおよびピトロネクチン様タンパク質の分子量やバンドの数が、種により多様であることが1つの問題である。また、ヒトピトロネクチンの分子量の問題点としては、SDS電気泳動で75 kDaである分子量が、一次構造より推定される分子量52 kDaよりもかなり大きいことがあった。これらの分子量の問題を解決することが、ピトロネクチンの統一分子像を見つけるカギになると思われた。分子量の問題は、①糖がポリペプチド鎖に結合しており、その含量が種により多様であること②ピトロネクチンの見かけの分子量がSDS電気泳動のゲル濃度によって変化するため、通常の分子量推定がピトロネクチンには当てはめることができないことの2つに起因する可能性が高かった。そこで、精製したピトロネクチンの糖鎖を切断し、ゲル濃度によらない分子量算定法のファーガソンプロットを行ってピトロネクチンの分子量を求めた。その結果、ピトロネクチンのポリペプチド鎖部分の分子量は40－57 kDaで、cDNAより推定される分子量とよく似た値となった。また、カルボキシ末端から5－13 kDaの位置にヒトピトロネクチンと同様のポリペプチド切断点があるとすると、動物ピトロネクチンはもともと50－57 kDaで合成されることになる。そして分子によって切断を受けるものと受けないものがあるため、分子量やバンドの数に多様性が生ずるのではないか。つまり、ピトロネクチンの動物種多様性は糖鎖部分とペプチド鎖切断の有無によるもので、基本的なポリペプチド鎖部分の構造は統一性をもつと推測された。, では、ピトロネクチン様分子として検出されたものも動物ピトロネクチンと同様の分子像を持つのだろうか。そこで、ピトロネクチン様タンパク質を検出できた最も原始的生物、粘菌のピトロネクチン様タンパク質について調べることにした。70 kDaの粘菌ピトロネクチン様タンパク質を粘菌のホモジネートの不溶性画分から、0.8 % TritonX-100で抽出し、抗体セファロースカラムクロマトグラフィー、へパリンセファロースカラムクロマトグラフィー、SDS電気泳動ゲルからの電気的抽出を経て精製した。精製された粘菌ピトロネクチン様タンパク質のアミノ末端アミノ酸配列は、高等動物のものとは相同性がみられなかった。しかし、この分子にはへパリン結合性やRGD配列依存性の細胞伸展活性があった。これらの結果より、粘菌ピトロネクチン様タンパク質は、動物ピトロネクチンと全く同じ分子像を持っているわけではないが、ピトロネクチン分子に非常に似た分子であると考えられた。\\r\\\ このようにピトロネクチンの大量精製法が確立し、分子像が明らかになってくると、他方ではピトロネクチンを細胞機能制御材料や医薬用血漿製剤に利用しようと考えるようになった。そのようなピトロネクチンの利用時に想定される処理条件として、煮沸やオートクレープによる滅菌がある。そこで、熱やオートクレープ処理によりピトロネクチンの細胞伸展活性がどの様に変化するかを調べた。その結果、ピトロネクチンは、100℃、10分の熱処理や121℃、1.2 kg / cm2、20分のオートクレープ処理によってもその細胞伸展活性を失うことはなく、その活性は未処理のものと同じ比活性を示し、RGD配列依存性であった。またオートクレープ処理によって、ピトロネクチンの分解と高分子量化が生じたが、この変化は還元剤の添加により部分的に抑制できた。以上のことから、ピトロネクチンを煮沸、オートクレープで滅菌し、医薬や細胞培養へと応用していくことが可能となった。, Vitronectin is a multifunctional glycoprotein in animal blood plasma. It promotes adhesion and migration of cells in an Arg-Gly-Asp(RGD) sequence dependent manner. Furthermore it modulates immune complement, blood coagulation and fibrinolytic systems. Vitronectin is also known to be a heparin-binding protein, and is instantly purified from human plasma by heparin affinity chromatography in the presence of urea. Although there have been many reports on vitronectin from human, there has been few reports from other animals and no report from primitive organisms.\\r\\\ To know a general structure and function of vitronectin, I tried to identify and characterize vitronectins in a variety of living organisms.\\r\\\ Animal plasma vitronectins can be purified from rabbit, mouse, rat, hamster, guinea pig, dog, horse, porcine, bovine, goat, sheep, chicken, and goose using heparin affinity column. The apparent molecular weights of these vitronectins range from 59 to 78 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1-3. Proteins reacting with anti-vitronectin antibo\dy were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almast all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.\\r\\\ I attempt to know the uniformity of animal vitronectin polypeptide. Purified animal vitronectins have similar amino terminal sequences. Furthermore, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptide were speculated to be synthesized de novo in size range of 50-57 kDa. Cell-spreading activity, heparin- and collagen-binding activities, which are buried through polypeptide region, are conserved among animal vitronectin. Therefore, animal vitronectins have uniformity in polypeptide and variety in sugar moiety.\\r\\\ Physarum is the most primitive organism producing vitronectin-like protein. To know the similarity of vitronectin-like protein in primitive organisms to animal vitronectins, the protein was purified to ho\mogeneity from Physarum microplasmodia by four steps: extraction from the insoluble cell debris, immunoaffinity chromatography, heparin affinity chromatography, and electroelution of preparative SDS-PAGE. The amino terminal sequence of Physarum vitronectin had no similarity to NH2-terminal sequences of animal vitronectins. Physarum vitronectin was efficient in mediating cell-spreading of BHK cells, which was specifically inhibited by RGD-containing synthetic peptides.\\r\\\ For application of animal vitronectins, I have investigated the heat and autoclave resistant properties of the cell-spreading activity of vitronectin. Vitronectin heated at 100 ℃ for 10 min or autoclaved at 121 ℃ at 1.2 kg / cm2 for 20 min retained the same cell-spreading activity as native vitronectin. In contrast, fibronectin and type I collagen treated in the same way lost its activity almost completely. GRGDSP remarkably inhibited the cell-spreading activity of native, heated, and autoclaved vitronectins. GRGESP did not inhibit the activity of the activity of native vitronectin, but unexpectedly partially inhibited the cell-spreading activity of the activity of both heated and autoclaved vitronectins. In SDS-PAGE, vitronectin heated at 100 ℃ migrated mainl\y as a monomer, but autoclaved vitronectin migrated at both the top and front of the gel instead of at the position of the monomer. The change in molecular size during the heat and autoclave treatments was partially prevented by adding 10 mM dithiothreitol to the protein solution., お茶の水女子大学人間文化研究科博士(理学)学位論文・平成5年3月23日授与(甲第29号)

Published
1993

40. Genome-wide Expression Analysis of Mouse Liver Reveals CLOCK-regulated Circadian Output Genes

Author

Oishi, Katsutaka, primary, Miyazaki, Koyomi, additional, Kadota, Koji, additional, Kikuno, Reiko, additional, Nagase, Takahiro, additional, Atsumi, Gen-ichi, additional, Ohkura, Naoki, additional, Azama, Takashi, additional, Mesaki, Miho, additional, Yukimasa, Shima, additional, Kobayashi, Hisato, additional, Iitaka, Chisato, additional, Umehara, Takashi, additional, Horikoshi, Masami, additional, Kudo, Takashi, additional, Shimizu, Yoshihisa, additional, Yano, Masahiko, additional, Monden, Morito, additional, Machida, Kazuhiko, additional, Matsuda, Juzo, additional, Horie, Shuichi, additional, Todo, Takeshi, additional, and Ishida, Norio, additional

Published
2003
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