片桐 昌直

The effects of 21-hydroxypregnenolone and related steroids such as deoxycorticosterone (DOC; 21-hydroxyprogesterone), cortisol, and corticosterone on progesterone 17 alpha-hydroxylase activity by steroidogenic cytochrome P450 c17 (CYP17) were investigated. 21-Hydroxypregnenolone contains a hydroxyl group at C-3 in the A cyclic hydrocarbon ring and a double bond at C-5 in the B cyclic hydrocarbon ring, whereas DOC, cortisol, and corticosterone contain a ketone group at C-3 and a double bond at C-4 in the A cyclic hydrocarbon ring. No marked inhibition was observed for DOC, cortisol, and corticosterone at 100 mu M concentration. Nonetheless, 21-hydroxypregnenolone exhibited competitive inhibition of progesterone 17 alpha-hydroxylation activity by CYP17 with a K-i value of 36.4 mu M. These results suggest that a hydroxyl group at C-3 in the A ring and a double bond at C-5 in the B ring in steroid hormones are important for the substrate recognition of CYP17.

2008年に科学技術振興機構が小学校教員を対象とした理科教育実態調査を行った。大阪府下の小学校教員の特徴や課題を明らかにするために,全国調査で使われた調査項目の一部を利用して同様の意識調査を実施した。その結果,理科の中で教える際の苦手意識が高い分野は,物理,地学,ICT教育が挙げられ,理科の知識や技能等の自己評価は,学習評価や自由研究などについての知識や技能が低いと自認している割合が高く,これらの結果は全国調査と同様の傾向を示す結果であった。一方,理科の指導方法に対する意識では,全国調査との違いが見られ,大阪府下の教員は,実験の手順や方法などの指導を重視する傾向が明らかとなった。A survey of awareness in science education to primary teachers in Japan was conducted by Center for Promotion of Science Education of Japan Science and Technology Agency in 2008. In order to characterize teachers of elementary school in Osaka, we investigated using this survey into an awareness of primary teachers in science education. A part of results showed that they have a consciousness weak about physics, earth science, and ICT education. Further, there were a lot of teachers who recognized to be scarce about the knowledge and skill about a method of study evaluation or instruction of independent research. These results were as well as the JST's survey. On the other hand, another data suggested that primary teachers in Osaka tend to regard instruction of procedure for an experiment as important.

海外における先進的な科学教育の現状を捉えるため,アメリカにおいて科学教育アウトリーチ活動が盛んである事情を分析するとともに,日本のスーパーサイエンスハイスクール事業を通じて行われている海外科学教育研修について調査を行った。さらに,附属高校天王寺校舎が実施しているスーパーサイエンスハイスクール事業の高校生海外研修に同行し,アメリカの大学が提供する発展的な内容を含む科学教育アウトリーチ活動,高校・大学間の連携に関する取り組み,先進的な科学教育に特化した高校での授業や課題研究の取り組み,および科学博物館での教育活動について調査を行った。The concern with science outreach programs has been growing for the last several years in Japan. In this paper, we take up outreach in America, are quite active, and study-tours of abroad for Super Science High School (SSH) students, participated in outreach programs of abroad universities and research institutes. We reported as a case of abroad study-tours of Tennoji High School attached to Osaka Kyoiku University.

Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to several oxygenase enzymes including cytochrome P450 (CYP). Genetic mutations in the POR gene have recently been identified and associated with an autosomal recessive genetic disease. In this study, V-max, K-m, and V-max/K-m, values of cytochrome c reduction and NADPH oxidation activities for R457H variant, histidine-tagged wildtype, and histidine-tagged E580Q were compared with those for wild-type. V-max/K-m, values of cytochrome c reduction for the R457H variant and histidine-tagged wild-type were 8% and 26%, respectively, of wildtype, whereas V-max/K-m values of NADPH oxidation for the R457H variant and histidine-tagged wild-type were similar to those for wild-type. The kinetic parameters of the histidine-tagged E580Q variant were similar to those for histidine-tagged wild-type, suggesting that E580Q mutation may be of minor importance in interindividual variation in drug response. These results suggest that R457H but not E580Q is essential for the deficiency of POR activities and that the histidine-tagged system would be inappropriate for POR function.

附属学校において実施されている科学教育活動の実践効果を捉えるため,PISA2006調査に採用されたアンケート項目を用いて,天王寺地区附属学校児童,生徒を対象に調査を行った。科学に関する認識や関心は,学年が進むにつれて,徐々に減少する傾向が示された。各学年の前期と後期の結果から,関心の向上の低下と回復が繰り返されていた。環境問題や社会における科学の価値など,小学校の時点から高い水準を示している項目は,学年が進むことによる減少は少ないことが示された一方,小学校の時点で低い水準を示した項目は,学年が進むにともなう減少の幅が大きかった。附属高校天王寺校舎で実施されているSSHカリキュラムに参加している高校生の科学に対する興味関心は,全学年を通じたトレンドと比較して,下げ止まる傾向が示された。The purpose of this research is to evaluate the trends of effects on attitudes to science of students of the science education curriculum of Tennoji elementary, junior high and high school attached to Osaka Kyoiku University. PISA 2006 Assessment was adapted to compare with the internal assessment results.1.All parameters about attitude for Science of students tended to decrease gradually as the grade in school advanced. 2.The higher-level parameters in elementary school held to high standard for the advanced grades.3.The low-level parameters in elementary school significantly decreased for the advanced grades.4.There was significantly the decrease-increase cycle of parameters about attitude for Science of each grade.

大阪教育大学は2009年10月に京都大学大学院理学研究科と協定を結び,すでに包括的協力協定を持つ大阪府教育委員会との三者共同で,大学院後期博士課程修了者および在学者の中から希望者を募って,高等学校の理数科教員として活躍できるよう支援する取り組み(高度専門型理系教育指導者養成プログラム:Excellent Science Teacher Training Program,略称ESTTプログラム)を立ち上げた。2011年6月には大阪大学大学院理学研究科との協定も成立し,四者共同の取り組みとなった。大学院で研究を行う中で知った研究の面白さや体験を中等教育の現場で生徒に直接伝え,次代を担う若人に知的な刺激を与えて学習意欲を高めてもらいたいとの考えからである。その高度な能力を活かし,理数科教育における中核教員として地域の科学教育力の向上に寄与してもらうこと,さらに,大阪教育大学の学生が受講者から知的な刺激を受ける効果も期待している。この取り組みの中での大阪教育大学の役割は,1)教員免許のための単位取得の機会の提供,2)現場体験のための支援,3)教員として必要な資質を育成・強化するためのセミナーの開催,などである。2010年4月に一期生を受け入れ活動を開始した。現在は手探りの試行的段階にあるが,このプログラムを始めたいきさつとその内容,および,今後の展望を紹介する。

Serum levels of 17-hydroxypregnenolone, dehydroepiandrosterone, 17-hydroxyprogesterone, and androstenedione were measured during the postnatal development of rats 1-14 weeks of age. A significant decrease in the serum levels of these steroids with increasing age was observed, using multiple regression analysis: 17-hydroxypregnenolone (beta = -1.56, S.E. = 0.25, P < 0.00001), dehydroepiandrosterone (beta = -0.43, S.E. = 0.07, P < 0.00001), 17-hydroxyprogesterone (beta = -2.51, S.E. = 0.45, P < 0.00001), and androstenedione (beta = -1.63, S.E. = 0.33, P < 0.00001). A sex-related difference was not found. The observed decline in the serum levels of the steroids was directly proportional to the previously reported decrease in mRNA expression and enzyme activity of cytochrome P450c17 in the rat liver. Yet, despite this decrease to undetectable levels in liver after 7-8 weeks, significant amounts of 17hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione were still observed in the rat serum. This may partly be due to the mRNA expression. of cytochrome P450c17 in tissues other than the liver, such as the testis and/or duodenum, after 4 weeks of age. Serum levels of pregnenolone, progesterone, and corticosterone in the developing rats were also examined. (c) 2005 Elsevier Inc. All rights reserved.

本学では,1994年より10年以上にわたり小学生高学年から高校生までの幅広い児童,生徒,延べ1,000名以上に対し,化学実験を体験する一日体験教室を化学系研究室合同で行ってきた。本稿では,これまでの経過,準備・実施体制,実施上の留意点などについて述べ,さらにこれまでの経験を通して,各学校段階での違いなどについても考えてみる。

In our previous study, we have investigated the serum levels of dehydroepiandrosterone (DHEA) in type 11 collagen (CII)-induced arthritis (CIA) mice. During the study, we found that in normal control mice, serum levels of DHEA in the latter half of the experimental period (13-16 weeks old) were significantly lower than those at the beginning of the experiment (10 weeks old). However, in CIA mice, such decreases were not observed by CH treatment. To examine the cause of the retention of DHEA during the development of arthritis in CIA mice in this study, 17alpha-hydroxylase/C17-20 lyase P450 (CYP17) mRNA expressions were measured by real time RTPCR and the CYP17 enzyme activities were investigated in the liver and testis on days 6, 13, 28 and 48 after CH treatment in DBA/1J mice. There were no significant differences of CYP17 expressions in the liver between control and CIA mice at each experimental day, while a significant increase of expression in the testis of CIA mice was observed on day 48. On the other hand, CYP17 enzyme activities on days 28 and 48 in testis microsome (Me) from the CIA mice were significantly higher than those of the control on the same day, while no significant differences of activities in liver Me were observed between the CIA and control mice. These findings suggested that the cause of the retention of DHEA on days 28 and 48 after CH treatment may he the increase of CVP17 expression and the enzyme activities in the testis.

Catalytic antibody 2B generated by immunization with N-methyl mesoporphyrin as hapten catalyzes the insertion of a cupric ion into mesoporphyrin. To identify amino acid residues essential for the catalytic activity, we studied effects of various amino acid-reactive reagents on the catalytic activity. The reagents reactive to Arg, Tyr and carboxyl-containing residues inactivated the antibody and mesoporphyrin protected notably the antibody from the inactivation. These results indicated that Arg, Tyr and carboxyl-containing residues are situated in or near the substrate-binding site of the antibody and that some of them would be essential for the catalytic activity. The modified At g and Tyr residues in the inactivation were quantified in connection with the residual activity. As the result, it was shown that three Arg and one Tyr residues are modified to lead the inactivation. Kinetic analysis indicated that the antibody loses the catalytic activity by modification of one carboxyl-containing residue. In order to find candidates for the modified residues, we performed modeling of the variable domain A the antibody. The model showed that the modified residues are Arg L54, Arg H94, Arg H95, Tyr L91 and Asp H96, and suggested that Arg H95, Tyr L91 and Asp H96 residues would stabilize the transition state of mesoporphyrin in the antibody-mediated reaction. (C) 2004 Elsevier B.V. All rights reserved.

The amino acid residues affecting the substrate specificity of human cytochrome P450 CYP2C9 and CYP2C19 for their metabolic activities were investigated using chimeras and mutant enzymes, which were constructed by replacing the corresponding residues. Although CYP2C19 showed nearly the same tolbutamide hydroxylase activity as CYP2C9, the activities for the CYP2C19( H99I) mutant and the chimeras that replaced residues 1-212 were much lower than those for CYP2C19. The activities of the CYP2C19( H99I) mutant and the chimeras that replaced residues 228-340 were lower than those for CYP2C19 toward S-mephenytoin, aminopyrine, and testosterone. These results suggest that residues in substrate recognition site (SRS) 3 and 4 are important for the substrate specificity, whereas His99 is important in the substrate binding of CYP2C19. For the 4'-hydroxylation of diclofenac, CYP2C9 had a lower K-m and a higher V-max than CYP2C19. Although the V-max values for the CYP2C9(1-288)/CYP2C19(289-490) chimera and the CYP2C9( I99H, V292A, F295L, I331V) mutant were comparable to those for CYP2C9, its Km value was comparable to that for CYP2C19. The V-max and K-m values for the CYP2C19(1-288)/ CYP2C9( 289-490) chimera were comparable to those for CYP2C19, and the activity by CYP2C9(1-230)/CYP2C19(231-490) chimera was negligible. These results suggest that the residues 292, 295, and/or 331 of CYP2C9 are essential for the recognition of substrate in CYP2C9 and that the residues of 231-288 including SRS 3 are important for the metabolizing capacity of CYP2C9.

Effect of nonylphenol on aminopyrine N-demethylase activity, a typical drug-metabolizing enzyme activity, by ten kinds of human hepatic cytochrome P450s (CYP) and on progesterone 17alpha-hydroxylase activity by steroidogenic CYP17 was investigated. When determined at 2 mm substrate concentration, nonylphenol (1 mm) most efficiently inhibited aminopyrine N-demethylation by CYP2C9 and CYP2C19, by 61% and 59%, respectively, followed by CYP2D6, CYP1A2, CYP2C18 and CYP2C8 (46-51%), whereas inhibition of the activities by other CYPs was less than 27%. Additionally, nonylphenol competitively inhibited diclofenac 4'-hydroxylation by CYP2C9 and S-mephenytoin 4'-hydroxyIation by CYP2C19 with K-i values of 5.3 and 37 muM, respectively. Furthermore, nonylphenol exhibited a competitive inhibition of progesterone 17alpha-hydroxylase activity by CYP17 with K-i value of 62 muM. These results suggest that nonylphenol inhibits human hepatic CYPs, especially CYP2C9 and CYP2C19, and steroidogenic CYP17 activities.

The metabolism of bisphenol A (BPA) was determined for 11 forms of human hepatic cytochromes P450 (CYPs) expressed in the yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli Additionally, the effect of BPA on the progesterone 17 alpha -hydroxylase activity of CYP17 was investigated. CYP2C18 catalyzed BPA metabolism most efficiently, followed by CYP2C19 and CYNC9. CYP2C9 and CYT2C18 exhibited the highest affinity (K-m,=3.9 muM) for BPA metabolism. The V-max, of CYP2C18 (8.10 nmol (.) min(-1) (.) nmol CYP-1) was 5 times higher than that of CYP2C9. Although the V-max of CYP2C19 was 1.5 times higher than that of CYP2C18, the affinity of CYP2C19 was 12 times lower than that of CYP2C9 and CYP2C18. Therefore the intrinsic clearance (V-max/K-m) of CYP2C18 was more than 5 times higher than that of CYP2C9 and CYP2C19. On the other hand, BPA exhibited a competitive-type inhibition of the progesterone 17 alpha -hydroxylase activity of CYP17 with a Ki value of 71 muM, whereas no metabolism of BPA by CYP17 was detected. These results suggest that BPA is mainly metabolized by the CYP2C subfamily in human liver, and that BPA inhibits human steroidogenic CYP17 activities.

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs, CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with K-i value of 97 mu M. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with K-i value of 113 mu M. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest V-max, followed by CYP2C19, 2D6 and 1A2, whereas the K-m for CYP2Cs, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the V-max/K-m for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b(5), whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

The maintenance of cytochrome P450s (P450s) and NADPH-cytochrome P450 reductase (P450 reductase) in the monolayer and spheroid cultures of hepatocytes from male rats was examined. The content of total P450 in monolayer culture decreased to almost none after 144 hr, whereas the level in spheroid culture remained within 6-13% of initial values during an incubation period of 144-192 hr. P450 2C11, a major P450 in male rat, in monolayer cells rapidly decreased in 144 hr, while the level in spheroid cells after 144 hr and 192 hr maintained 25% and 15%, respectively, of initial level. On the other hand, P450 2A1 and P450 2E1 in both monolayer and spheroid cells rapidly decreased. P450 reductase in both cells showed a gradual decline reaching a level of 43-44% of the initial level at 96 hr, and remained within 16-17% of the initial value during an incubation period of 192 hr. These results indicate that P450 2C11 in spheroid cells maintained more stable than in the monolayer cells, and that P450 reductase in both cultures declined only moderately, compared with P450s.

We investigated the expression of NADPH-cytochrome P450 reductase (P450 reductase) in various tissues from rabbits. The size of RNA from liver, lung, and kidney of rabbit was compared. Northern hybridization with total RNA indicated that the P450 reductase mRNA in liver was 21 S size, which corresponds to 2.3 kb, and there was no difference in the size of P450 reductase mRNA between liver and extrahepatic tissues. The tissue-specific expression of P450 reductase was investigated by measuring its activity and mRNA content in these tissues of rabbit. To compare the levels of the reductase mRNA between rabbit tissues, the amount of b-actin mRNA for normalization was used. A good correlation was observed between the mRNA and the enzyme activities of P450 reductase in rabbit liver, lung, and kidney. The increase in the activity by phenobarbital (PB) treatment was not observed in any of the tissues. The amount of P450 reductase mRNA in the liver was slightly increased and that in the kidney was not changed, while the level of P450 reductase mRNA in the lung was induced approximately 2-fold. These results suggest that P450 reductase mRNA in rabbit tissues, which is transcribed from an identical gene, is translated to an identical protein molecule, and that the response of P450 reductase mRNA and protein to induction by PB in each tissue is different among the different tissues of the rabbit.

Human cytochrome b(5) has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b(5). Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b(5). The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b(5) plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b(5)/P450 ratio, 17 alpha-hydraxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b(5) in the human testis, both the Delta 4 and the Delta 5 steroidogenic pathways can lead to testosterone production. (C) 1995 Academic Press, Inc.

The activity of protoporphyrinogen-oxidizing enzymes was found not only in crude etioplast and mitochondrial fractions but also in the soluble fraction of tobacco cell lines. Approximately 90% of the total activity was found in the soluble fraction of the SL cell line. A protoporphyrinogen-oxidizing enzyme was purified from the soluble fraction of SL by chromatography on CM-Toyopearl, hydroxyapatite, and HA-1000 columns. The purified enzyme has a molecular weight of approximately 48,000 on SDS-polyacrylamide gel electrophoresis. Apparent K-m and V-max values of the purified enzyme for protoporphyrinogen IX were 78.9 mu M and 1.3 mu mol/mg protein/min, respectively. The purified enzyme utilized uroporphyrinogen I and coproporphyrinogen I as substrates. The protoporphyrinogen-oxidizing activity of the purified enzyme was not inhibited by herbicides that inhibit protoporphyrinogen oxidase. The purified enzyme contained a heme and showed peroxidase activity toward guaiacol and pyrogallol. On the other hand, peroxidases commercially available showed the protoporphyrinogen-oxidizing activity. Based on these results, the soluble protoporphyrinogen-oxidizing enzyme in tobacco cultured cells seemed to be a kind of peroxidase. The soluble protoporphyrinogen-oxidizing enzyme with herbicide resistance may play an important role in the oxidation of protoporphyrinogen IX which accumulates out of the site of heme and chlorophyll biosynthesis in the herbicide-treated plants. (C) 1994 Academic Press, Inc.

1. By means of an enzyme immunoassay, the contents of D-amino acid oxidase (DAO) were determined in kidney, liver, cerebellum and lung of hog, but the oxidase was not detectable in heart or cerebrum. 2. The oxidases in kidney, liver and cerebellum of hog were indistinguishable as regards immunoreactivity toward anti-hog kidney DAO antibody, specific activity and molecular weight. 3. The oxidases in rat and dog kidneys immunochemically cross-reacted with anti-hog DAO antibody. 4. The overall structure of the hog oxidase was more similar to that of the dog enzyme than that of the rat, while the structure around the catalytic site of the hog oxidase was more similar to that of the rat oxidase than that of the dog enzyme. 5. On immunoblot analysis, two forms of the oxidase were detected in extracts of hog, rat and dog kidneys.