Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells.

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).</p

Chemo-enzymatic hybrid process for production of monatin, a high intensity sweetener

Monatin, 4-hydroxy-4-(3-indolylmethyl)-glutamic acid, is a naturally occurring sweet amino acid isolated from the plant Sclerochiton ilicifolius, found in South Africa [1]. Monatin has two asymmetric centers at C2 and C4, the (2R,4R)-monatin isomer has been found to be the sweetest among its four stereoisomers. It is 2700 times sweeter than sugar and has a clean taste like sugar. Because of these properties, (2R,4R)-monatin has been expected as an new high-intensity sweetener [2]. However, industrial production process of (2R,4R)-monatin using inexpensive raw materials has not been established owing to the difficulty for optically specific synthesis. Here, we report a chemo-enzymatic hybrid process for production of (2R,4R)-monatin from l-tryptophan. In the steps of enzymatic reaction from l-tryptophan, l-amino acid deaminase and aldolase were used for production of 4-(Indole-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG) with pyruvic acid as co-substrate. The keto-form of (2R,4R)-monatin, (R)-IHOG, was specifically synthesized by using R-specific aldolase from Shingomonas sp. in the second reaction. In the next chemical reaction steps, (R)-IHOG was converted to the oxime form, reduced to (2R,4R) and (2S,4R)-monatin, and (2R,4R)-monatin salt was obtained from optical resolution by crystallization. By the combination of epimerization and crystallization, (2R,4R)-monatin was obtained specifically from the mixture of diastereomers. In this study, we established an efficient production process for (2R,4R)-monatin using both chemical and enzymatic reactions, and a large amount of (2R,4R)-monatin was prepared by the bench-scale production. Please click Additional Files below to see the full abstract

磁場を利用した電極反応の促進に関する研究

金沢大学工学部基礎実験として,円柱陰極および球陰極を使用し,鉛直方向および水平方向に静磁界を印加した場合の限界電流密度(物質移動速度に対応)を測定した.その結果,磁界印加の方向性,陰極形状およびその方向によって,磁束密度が物質移動速度の促進に様々な異なる影響を与えることを確認した.次に,データを相関する実験式を纒るため,電磁流体力学および移動現象論に関わる諸微分方程式を正規化することによりパラメータを探索し,レイリー数,シュミット数,ローレンツ数の諸無次元数が関与することを見いだし,これらのべき乗の積の表現式でデータがうまく相関できることを示した.なお,ローレンツ数は本研究で始めて使用を提唱する無次元数である.実験より1Tの静磁場印加により電極反応(物質移動)速度が2〜3倍に促進されることが明かとなった.以上の基礎実験結果を基礎に,円柱状陰極を用いてパルス磁界を用いて促進効果を検討し,以下の結果を得た.磁界より発生させられた電磁流体力学的流れは,磁界の除去後30〜40秒の緩和時間を伴って自然対流状態へ遷移する.よって,1Hz以上の高周波数の磁界の印加は効果が乏しい.0.2Hz以下の磁界による促進効果は静磁場の場合より僅かに大きいが,磁界の一周期の平均磁束密度を代表磁束密度と考えれば,ほぼ静磁場と同一の相関式で纒められる.この成果は,平成5年10月の化学工学会秋季大会で口頭発表した.交番磁界を用いた実験では,常時磁界を印加している事になるにも関わらず,電極反応の促進効果は,静磁場程大きくはなく,主たる磁場方向の印加時間に比例することを見いだした.加えて,局所物質移動速度を測定した結果,円柱の周方向に均一な物質移動状態を交番磁界の印加により実現できることを示した.これは,電着による表面処理の高度化につながるものと期待される.この成果は,平成6年7月化学工学会で口頭発表する.研究課題/領域番号:04650847, 研究期間(年度):1992出典：研究課題「磁場を利用した電極反応の促進に関する研究」課題番号04650847（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/grant/KAKENHI-PROJECT-04650847/）を加工して作

Hereditary cataract of the Nakano mouse: Involvement of a hypomorphic mutation in the coproporphyrinogen oxidase gene

The Nakano cataract (NCT) is a recessive disorder in the mouse linked to the nct locus on chromosome 16. In this study, we positionally cloned the critical gene in the nct locus. Herein, we report that cataracts in the BALB/c-nct/nct mouse are caused by a hypomorphic mutation in the coproporphyrin oxidase gene (Cpox), encoding the enzyme responsible for catalyzing oxidative decarboxylation of the heme precursor, coproporphyrinogen III, in the heme biosynthetic pathway. BALB/c-nct/nct mice are homozygous for a G to T nucleotide substitution in the Cpox gene, which results in a p.R380L amino acid substitution in the CPDX protein. The CPDX isoform with the p.R380L substitution retained only 15% of the activity of the wild type isoform. BALB/c-nct/nct mice had excessive accumulation of coproporphyrin HI in the lens. The NCT phenotype was normalized by the introduction of a wild type Cpox transgene. The mechanisms by which impairment of CPDX leads to lens opacity in the NCT are elusive. However, our data illuminate a hitherto unanticipated involvement of the heme biosynthesis pathway in lens physiology.ArticleEXPERIMENTAL EYE RESEARCH. 112:45-50 (2013)journal articl

Implantable pneumatically actuated microsystem for renal pressure-mediated transfection in mice.

In vivo transfection is an important technique used in biological research and drug therapy development. Previously, we developed a renal pressure-mediated transfection method performed by pressing a kidney after an intravenous injection of naked nucleic acids. Although this is a useful method because of its safety and wide range of applications, an innovative approach for performing this method without repeatedly cutting open the abdomen is required. In this study, we developed an implantable microsystem fabricated by Micro-Electro-Mechanical Systems (MEMS) technologies for renal pressure-mediated transfection. The system consists of a polydimethylsiloxane pneumatic balloon actuator (PBA) used as an actuator to press the target kidney. The PBA of the implanted microsystem can be actuated without opening the abdomen by applying air pressure from outside the body to the pressure-supplying port via a needle. We successfully performed renal pressure-mediated transfection using the newly developed system when the implanted system was activated at 60kPa for 10s. This is the first report of an implantable MEMS-based microsystem that demonstrates in vivo transfection to a kidney using naked plasmid DNA

Enhancement of electrolytic mass transfer around spheres by applying static magnetic fields

金沢大学大学院自然科学研究科エコサイクルシステム金沢大学工学部The effect of applying a static magnetic field on mass transfer rate in diffusion-controlled electroreduction was studied experimentally around single spheres of diameters 8 to 14 mm under the condition of laminar natural convection. The electrolytic solution of the system K"SUB 3" Fe(CN)"SUB 6" -K"SUB 4" Fe(CN)"SUB 6" with a supporting electrolyte was employed and the magnetic field was applied to the cathode in the horizontal or vertical direction and up to 336 mT in flux density. By applying the magnetic field in every direction, the mass transfer rate was enhanced more than 50% at the highest magnetic flux density, compared to the simple natural convection case. (from Authors)

Deoxyribonuclease I sensitivity of DNA replicated in permeable mouse sarcoma cells.

To study chromatin structure at the sites of DNA replicated in permeable cells, deoxyribonuclease I (DNase I) sensitivity of newly replicated DNA in permeable mouse sarcoma cells was compared with that of newly replicated DNA in intact cells. About 35% of the DNA replicated in permeable cells was hypersensitive to DNase I, and the remaining DNA showed the same DNase I sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in intact cells, and was close to that of DNA replicated in the presence of cycloheximide. The sensitivity of DNA pulse-labeled with [3H]deoxythymidine triphosphate by replication in permeable cells was reduced significantly by chasing with cold deoxythymidine triphosphate. The present results suggest that chromatin structure at the sites of DNA replicated in permeable cells is similar to that at the sites of DNA replicated in living cells in the absence of protein synthesis, and that some structural change (possibly toward the maturation) of newly replicated chromatin occurs after the DNA replication in permeable cells.</p

Bleomycin-induced DNA synthesis in a cell-free system using a permeable mouse sarcoma cell Extract.

To investigate factors involved in excision repair DNA synthesis, a soluble extract was prepared from permeable mouse sarcoma (SR-C3H/He) cells by homogenization and ultracentrifugation. DNA synthesis measured by using native calf thymus DNA as the template-primer and the extract as the polymerase source showed low activity. The DNA synthesis was enhanced more than ten-fold by the addition of an appropriate concentration of bleomycin, a radiomimetic DNA-damaging drug. Using selective inhibitors of DNA polymerases, it was shown that the DNA polymerase involved in the bleomycin-induced DNA synthesis was DNA polymerase beta. In addition to DNA polymerase beta, an exonuclease which converts bleomycin-damaged DNA into suitable template-primers for repair DNA synthesis appeared to be present in the permeable cell extract.</p

Environmental Factors and Seasonal Influenza Onset in Okayama City, Japan: Case-Crossover Study

Seasonal influenza infection is a major challenge in public health. The term "seasonal influenza" refers to the typical increase in the number of influenza patients in the winter season in temperature zones. However, it is not clear how environmental factors within a single flu season affect influenza infection in a human population. Therefore, we evaluated the effects of temperature and humidity in the 2006-7 flu season on the onset of seasonal influenza using a case-crossover study. We targeted patients who attended one pediatric clinic in Okayama city, Japan and who were diagnosed as being infected with the seasonal influenza virus. Using 2 references (time-stratified and symmetric bidirectional design), we estimated the effects of average temperature and relative humidity from the onset day (lag0) to 10 days before (lag10). The total number of subjects was 419, and their onset days ranged from 26 December 2006 to 30 April 2007. While the onset was significantly associated with lower temperature, relative humidity was not related. In particular, temperatures before the 3-day incubation period had higher-magnitude odds ratios. For example, the odds ratio and 95% confidence interval for average temperature at time lag 8 was 1.12 (1.08-1.17) per 1.0℃ decrease. Low environmental temperature significantly increased the risk of seasonal influenza onset within the 2006-7 winter season