イニシヤトリンとそのインヒビターによる精子運動制御機構の解明

金沢大学理学部"イニシャトリン"は、カイコガ前立腺から分泌されるセリン型エンドペプチターゼで、蛋白質のアルギニンのC側でポリペプチド鎖を切断する。リジン及びアルギニンのC側で切断する哺乳類膵臓由来のトリプシンは、作用の類似を利用して、イニシャトリンのモデルとして代換え出来る。イニシャトリンは、カイコガの無核精子のみならず、他の蛾の無核精子、そしてバッタの有核精子の運動性を獲得させる。また、カイコガ有核精子束を解離し、アルギニン・カルボキシペプチダーゼの前駆体を活性化する。それと共に、アルギニン分解カスケードを作動させ、精子にエネルギーを与える。この精製標品は、前立腺では30kDaで、少量の29と26kDaのも見出された。前立腺分泌液を含む精液で雌交尾襄内に構築される、精包由来精製イニシャトリンは29と26kDaであった。これらは、前立腺由来30kDa標品を免疫原とする抗体に基く、免疫学的解析によって確認された。イニシャトリンの極めて強い自己分解性を考え合わせると、前立腺では、まず30kDaイニシャトリンが分泌され、それが29kDaと26kDaに自己分解するように思われる。精包由来精製29kDaイニシャトリンは、無核精子賦活作用が弱いが、前立腺由来30kDaイニシャトリンは強く、N末20以外のアミノ酸配列がこの作用に関係する事を示唆する。その上、このイニシャトリンは、上記"イニシャトリン"としての条件を全て満足した。前立腺にイニシャトリンと共に存在するそのインヒビターは、約1万の分子量を待ち、耐熱性を有する。恐らく細胞保護のため、イニシャトリンと結合した状態で細胞内に存在し、分泌後、射精によって、イニシャトリン分子から離れ、活性化させると思われる。イニシャトリンを貯精襄液内精子と共に雌交尾襄内へ注入すると、卵を受精せしめる事が出来た。イニシャトリンは単なるエンドペプチダ-ではなくて、受精に不可欠の因子である。Initiatorin, an endopeptidase localized in the glandula (g.) prostatica of the silkmoth, Bombyx mori, triggers the arginine degradation cascade, a specific energy-yielding system for spermatozoa. This protease activates the precursor of arginine carboxy-peptidase which acts at the second step of the arginine degradation cascade by producing free arginine from the Arg-peptide splitted by initiatorin from a substrate protein. This multi-functional enzyme also initiates the dissociation of eusperm bundles and motility of apyrene sperm. The secreting feature from cells of the g. prostatica to its lumen was immunohistologically observed with antiserum which had been prepared from a purified initiatorin preparation. Initiatorin was isolated from the two organs, the g. prostatica and spermatophore, separately. In the former purification procedures hydrophobic interaction chromatography and cation exchange chromatography were applied, whereas affinity chromatography on p-aminobenz- amidine was in the latter. These obtained initiatorins were assigned by their N_-bezoylarginine ethyl ester-hydrolysing activity and also immunoblot after development of SDS-acrylamide gel-electrophoresis. Two types of initiatorins of molecular weights of 29 and 26kDa were isolated from the spermatophore, while three types of 30, 29 and 26kDa were from the g. prostatica. Ten N-terminal amino acid sequence of these initiatorins were identical. Prostatic 30kDa-initiatorin showed the strong induction of apyrene motility, but spermatophore 29kDa initiatorin did weak induction. 30kDa initiatorin satisfied the other three conditions as the multi-functional enzyme. Considered with highly strong self-degradation of initiatorin, 30kDa initiatorin produced in the cells of the g. prostatica must degrade to other initiatorins with smaller molecular weights in the g. prostatica and also in the sprmatophore. The initiatorin inhibitor localized in the g. prostatica seems to have an effect to protect cells to avoid the attack from this drastic protease. After unbinded from this inhibitor on ejaculation, initiatorin apparently "activated" causes many various reactions for sperm maturation. Fertilized eggs were laid, when a mixture of initiatorin and the content of the vesicula seminalis containing eusperm bundles and apyrene sperm was injected to the bursa copulatrix of virgin female moths instead of the spermatophore. Therefore, initiatorin is the indispensable factor for sperm maturation which causes the exact fertilization and fecundity.研究課題/領域番号:07640899, 研究期間(年度):1995 – 1996出典：研究課題「イニシヤトリンとそのインヒビターによる精子運動制御機構の解明」課題番号07640899（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-07640899/076408991996kenkyu_seika_hokoku_gaiyo/）を加工して作

蚕蛾精子の運動性獲得機構の解明,蚕蛾人工受精への挑戦

金沢大学理学部カイコガ精子には二型ある。いずれも精巣で作られた後、貯精嚢にて射精まで待機するが、無核精子、有核精子束のいずれも動かない。無核精子の運動性獲得因子は、雄生殖輸管の末端に位する前立腺に局在することが、まず突き止められた。次いで、この因子が、前立腺から分泌されるセリンプロテアーゼ型エンドペプチダーゼ、イニシャトリンであることが確認された。この酵素は、アルギニンのC側で、ペプチド鎖を切断する。SDS-PAGEによって、分子量は30kDaと測定された。それから分離されたイニシャトリン標本より抗体を作製した。交尾前の活性上昇は、mRNAの翻訳によって生ずる酵素分子の増加に他ならない。この酵素を、雌交尾嚢に作らせた精包から、BAEEを基質としてassayを行い、HPLCで精製した。極めて不安定で4種のisoformが得られた。その中の2種は29KDa、他の2種は26KDaで、何れもBAEEase活性を表すが、無核精子の賦活能は弱く、30KDa-イニシャトリンの部分解物ではないかと考えられる。29KDaの分子量を持つI型のシーケンスをN末から20のアミノ酸について、GENETYXによる検索を行った。この酵素は、8種の哺乳類のエンドペプチダーゼとの高い相同性を示した。次に、出発材料として雄前立腺を用い、アフィニティカラムを備えたFPLCで精製した。2つのピークが得られ、それぞれ、30KDaと29KDaであった。このKDa-イニシャトリンは、少量で無核精子を賦活した。ヤママユガでも、イニシャトリン類似のエンドペプチダーゼが見出され、無核精子を活性化し、カイコガとは相互互換性も見出された。直し目の有核精子の運動性獲得機構も、カイコガおよびヤママユガ等、鱗し目の無核精子と同一であった。The silkmoth, Bombyx mori(Bombycidae) produces two types of spermatozoa, apyrene spermatozoa and eupyrene spermatozoa that fertilize eggs. In the vesicula seminalis of male moth, these spermatozoa are immotile. We found that the apyrene sperm-activating factor (AAF) is secreted from the glandula prostatica of the male reproductive tract, and that AAF is initiatorin, an endopeptidase of serine-protease type that splits proteins at the arginine residues on the C-side. Molecular weight of this enzyme was determined as 30KDa with SDS-PAGE.Antiserum was prepared from a band of this PAGE corresponding to initiatorin. Immunological analysis demonstrated that the rise in this enzyme activity at the emergence results in an increase in the enzyme molecules produced by translation of mRNA.Initiatorin was purified using HPLC from the spermatophore formed in the female bursa copulatrix. The enzyme activity was tested with BAEE as a preferred substrate of initiatorin. This protease is very heat-labile. After purification four isoforms, two 29KDa and two 26KDa were obtained. Although they have high specific activities of BAEEase (200-fold purified), their abilities inducing sperm motility is relatively not strong. They may partially degraded products of the true 30KDa-initiatorin found in the glandula prostatica. Twenty amino acids of a purified 29KDa preparation, initiatorin I was sequenced from the N-terminal. Examination with GENETYX showed that this had high homologies with eight endopeptidases of various mammalian tissues. The 30-KDa initiatorin preparation purified from the glandula prostatica showed a high motility-inducing activity. In a wild silkmoth, Antheraea yamamai(Saturniidae), initiatorin-like endopeptidase was also found. This enzyme activated Antheraea-sperm as well as Bombyx-sperm. The identical mechanism of acquisition of sperm motility was also found in the orthopteran insects.研究課題/領域番号:03455026, 研究期間(年度):1991 – 1993出典：研究課題「蚕蛾精子の運動性獲得機構の解明,蚕蛾人工受精への挑戦」課題番号03455026（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-03455026/034550261993kenkyu_seika_hokoku_gaiyo/）を加工して作

Individual Radiation Exposure Dose Due to Support Activities at Safe Shelters in Fukushima Prefecture

Immediately after the accidents in the nuclear power stations in Fukushima on March 11, the Japanese Government ordered the evacuation of the residents within a 20-km radius from the station on March 12, and asked various institutions to monitor the contamination levels of the residents. Hirosaki University, which is located 355 km north of Fukushima City, decided to send support staff to Fukushima. This report summarizes the results of the exposure of 13 individual teams from March 15 to June 20. The support teams surveyed more than 5,000 people during this period. Almost all subjects had external contamination levels of less than 13 kcpm on Geiger-Müller (GM) survey meter, which is categorized as “no contamination level.” The 1st team showed the highest external exposure dose, but the 4th team onward showed no significant change. Subsequently, the internal radiation exposure was measured using a whole body counter that indicated undetectable levels in all staff members. Although the measured external radiation exposure dose cannot have serious biological effects on the health of an individual, a follow-up study of the residents in Fukushima and other regions where the radioactive material has spread will be required for a long time

Radiation dose reduction efficiency of buildings after the accident at the Fukushima Daiichi Nuclear Power Station.

Numerous radionuclides were released from the Fukushima Daiichi Nuclear Power Station (F1-NPS) in Japan following the magnitude 9.0 earthquake and tsunami on March 11, 2011. Local residents have been eager to calculate their individual radiation exposure. Thus, absorbed dose rates in the indoor and outdoor air at evacuation sites in the Fukushima Prefecture were measured using a gamma-ray measuring devices, and individual radiation exposure was calculated by assessing the radiation dose reduction efficiency (defined as the ratio of absorbed dose rate in the indoor air to the absorbed dose rate in the outdoor air) of wood, aluminum, and reinforced concrete buildings. Between March 2011 and July 2011, dose reduction efficiencies of wood, aluminum, and reinforced concrete buildings were 0.55 ± 0.04, 0.15 ± 0.02, and 0.19 ± 0.04, respectively. The reduction efficiency of wood structures was 1.4 times higher than that reported by the International Atomic Energy Agency. The efficiency of reinforced concrete was similar to previously reported values, whereas that of aluminum structures has not been previously reported. Dose reduction efficiency increased in proportion to the distance from F1-NPS at 8 of the 18 evacuation sites. Time variations did not reflect dose reduction efficiencies at evacuation sites although absorbed dose rates in the outdoor air decreased. These data suggest that dose reduction efficiency depends on structure types, levels of contamination, and evacuee behaviors at evacuation sites

Evacuation sites examined for absorbed dose rates in the indoor and outdoor air in the Fukushima Prefecture.

<p>Evacuation sites were 21–64 km from F1-NPS, and the buildings were aluminum-related (blue), reinforced concrete (yellow), and wooden (green) constructions. The map was generated using GPS data from each site. Markers of G1–G3 are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101650#pone-0101650-g002" target="_blank">Figure 2</a>.</p

Environmental half-lives of outdoor radioactive molecules at each evacuation site.

<p>Environmental half-lives (HFs) at cities of evacuation sites G1–G3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101650#pone-0101650-g001" target="_blank">Figure 1</a>) were calculated using the least squares method with the obtained data;</p>a<p><i>p</i><0.01 vs. early stage (15–31 March) of HFs at G2 and G3;</p>b<p><i>p</i><0.05 vs. early stage of HFs at G3;</p>c<p><i>p</i><0.05 vs. delay stage (April–July) of HFs at G3, and <i>^dp</i><0.05 vs. delay stage of HFs at G3; by Tukey–Kramer's test.</p

Variations in absorbed dose rates in the air immediately after the accident at F1-NPS.

<p>Dose rates in air were analyzed and calculated until July 2011 at G1, G2, and G3 sites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101650#pone-0101650-g001" target="_blank">Figure 1</a>). G1–G3 sites were measured 11, 21, and 10 times, respectively, on different days. The Nuclear Regulation Authority (NRA) data for each city are shown with lines (red: Fukushima city, blue: Koriyama city, light blue: south area of Minami-soma city), and values measured by our team are shown with circles. Solid and open circles represent outdoors and indoors, respectively.</p