Development of thermosensitive polymer-conjugated enzyme for repeated use.

温度感受性ポリマー誘導体を作製し、アルカリホスファターゼ（ALP）と結合させることによって、反復利用が可能なALPの開発を試みた。温度感受性ポリマー結合ALPは、反応混合液の温度を37℃以上に温度を上げることによって、簡単に他の成分から分離回収することができ、回収後も約80％の活性を保持していることが明かとなった。For the purpose of repeated use of enzyme, alkaline phoshatase congugated with the reactive derivative of thermosensitive polymer was developed. This novel enzyme was precipitated immediately and recovered from reaction mixture, when the solution temperature was raised to 37℃. Recovered enzyme retained 80% activity compared to that of untreated one

ヒスチジンタグを持つホスファカンコア蛋白の大腸菌での発現と精製

Specific regions of core protein of phosphacan, one of the chondroitin sulfate proteoglycans, were expressed as fusion proteins with histidine-tag (His-tag) in Escherichia coli (E.coli) and were affinity purified using nickel-nitrilotriacetic acid (Ni-NTA) matrix. cDNA fragments encoding amino acid residues 343-446 (P3) and 1-340 (P4) of phosphacan core protein were amplified by polymerase chain reaction from E18 rat brain mRNA as template. The amplified products were subcloned into pQE30 vector and were introduced into E.coli strain M15 [pREP4] for the expression. The His-tagged fusion proteins were expressed by cultivating the transformants at 37℃ for 5h in the presence of 1mM IPTG. His-tagged P3 fusion protein (His-P3) was expressed as soluble form, and was purified using Ni-NTA matrix. His-tagged P4 fusion protein (His-P4) which was sequestered into insoluble inclusion bodies was treated with 8.0M urea to solubilize, and then was purified under denaturing conditions.コンドロイチン硫酸プロテオグリカンの一つであるホスファカンのコア蛋白の特定領域を,ヒスチジンタグ(His-tag)を持つ融合蛋白として大腸菌内で発現させニッケル-ニトリロ3酢酸(Ni-NTA)アフィニティ担体を用いて精製した｡ホスファカンコア蛋白のアミノ酸残基343-446(P3)及び1-340(P4)に相当するcDNA断片を,胎性18日目のラット脳由来のmRNAを鋳型としたPCRによって増幅した｡増幅された断片は発現ベクターpQE30に組み込まれ,これで大腸菌(M15[pREP4])を形質転換した｡His-tag融合蛋白の発現は形質転換株を1mM IPTG存在下で37℃,5時間培養することによって行われた｡His-tagged P3融合蛋白は可溶性蛋白質として発現し,Ni-NTA担体を用いて精製された.His-tagged P4融合蛋白は不溶性の封入体を形成したが,8M尿素によって可溶化され,変性条件下で同様に精製された

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

Enzyme Immunoassay for Rabbit C-reactive Protein

Sensitive enzyme Immunoassay method specific for rabbit C-reactive protein was established. This was based upon specific Ca(2+)-dependent binding profile of C-reactive protein for some compounds containing phosphorylcholine moiety intramolecularly. By this method, more than 0.001μg of rabbit C-reactive protein was detectable. Specific binding of C-reactive protein for p-aminophenylphosphorylcholine immobilized on solid phase was inhibited by either addition of EDTA or phophorylcholine analogues, effectively. It may be useful to study possible roles of C-reactive protein in inflammatory regions.ウサギC-反応性蛋白質を感度良く測定できる酵素免疫測定法を開発した。本法は、C-反応性蛋白質が分子内にホスホリルコリンを含有する種々の化合物に対してCa(2+)依存的な結合活性を示すことに基づいており、本法によって0.001μg以上のC-反応性蛋白質の検出が可能となった。固相に結合させたp－アミノフェニルホスホリルコリンへのC-反応性蛋白質の特異的な結合活性はEDTAやホスホリルコリン誘導体を添加することによって効果的に消失した。本法は炎症部位でのC-反応性蛋白質の役割を明らかにする上で有効な手段となるものと考えられる

Alteration of thermostable phosphatase activity after hydrophobic chromatography

耐熱性ホスファターゼを含んだBacillus stearothermophilus 粗酵素試料を、リソースIsoによる疎水性クロマトグラフィにかけ分離を行った。1.5M→0M 硫酸アンモニウムの直線逆濃度勾配によって溶出を行ったところ、ホスファターゼは不活性な形で溶出され、これは硫酸アンモニウムによる濃度依存的阻害に起因することが判明した。ホスタファーゼの反応混合液に種々の濃度の硫酸アンモニウムを添加したところ、0.15Mの硫酸アンモニウム存在下で約80％の阻害が認められた。加えて、この阻害作用は単に硫酸アンモニウムの添加によってpHが酸性側に傾くことによるものではないことも明らかとなった。Thermostable phosphatase partially purified from thermophilic bacteria, Bacillus stearothermophilus, was chromatographed on Resource Iso hydrophobic resin. When linear reverse gradient elution with 1.5 M → 0 M ( NH(4))(2)SO(4) was performed, phosphatase was found to be eluted as latent form, which revealed dose-dependent inhibitory effect of (NH(4))(2)SO(4) on phosphatase. When various concentrations of (NH(4))(2)SO(4) were added into phosphatase reaction mixture, about 80% inhibition was observed in the presence of 0.15 M (NH(4))(2)SO(4). Acidification by adding (NH(4))(2)SO(4) was not responsible for this inhibition, because addition of (NH(4))(2)SO(4) solution which pH was previously adjusted to 9.0 showed same inhibitory effect

CONTINUOUS MEASUREMENTS OF COSMIC-RAY INTENSITY WITH MULTI-DIRECTIONAL MUON TELESCOPE 50 M.W.E. UNDERGROUND AT MATSUMOTO

A multi-directional muon telescope having an area of 8m² was constructed in a tunnel with a vertical depth of approximately 50 m. w. e. at Matsumoto. Continuous measurements of cosmic-ray intensity have been made since April 7, 1971. A preliminary report is presented concerning the underground site, experimental apparatus, and its arrangement, as well as some examples of experimental data obtained, and analyzed results of cosmic-ray daily variation. Counting-rates are 6 x 10⁴/hr of vertical component for 8 m² area at a depth of ～50 m. w. e., and 3.4 x 10⁴ /hr of N-component at ～42 m. w. e., 0.85 x 10⁴ /hr of S-component at ～92 m. w. e. for each of 6 m² area, 1. 1 x 10⁴ /hr of E-component at ～65 m. w. e., and 1. 55 x 10⁴ /hr of W-component at ～53 m.w.e. for each of 4m² area, inclined at 40° to the vertical. It is found that the observed phases of diurnal and semidiurnal vectors for five components are indicative of the extraterrestrial nature of the daily variation. The observation thus may provide an important information on the modulation of cosmic-ray intensity variation.Article信州大学教養部紀要. 第二部, 自然科学 6: 9-30(1972)departmental bulletin pape

Utilization of a serum-free primary culture of cortical neurons by using cyclodextrins in neurobiological research

神経生物学的研究における基本的分析系の確立を目的として、シクロデキストリン（CD）を用いたラット大脳皮質神経細胞の初代培養を試みた。β-およびγ-CDは、無血清培地（ダルベッコ改変MEM/ハム培地）中で胎生16および18日目ラットの神経細胞を11日以上10％胎児ウシ血清を加えた培地中と同じ程度に生存させたが、α-CDには生存維持効果が無かった。β-CDはγ-CDより安定した生存維持効果を示したが、胎生21日目ラットの神経細胞を用いた場合は有意に生存率が低下し、新生児ラットでは生存維持効果が無かった。β-CDを用いた無血清培養では10％血清培地中と比べて神経突起の伸展が悪かったが、ときに顕著な突起伸展がみられ、これはCD分子に取り込まれた生理活性物質の作用と考えられた。また、β-CDを用いた無血清培養を利用してラット脳から精製したコンドロイチン硫酸プロテオグリカン（CSPG）の作用を検討し、CSPGがグルタミン酸による神経細胞死を防止すること、弱いながら培養神経細胞の生存を維持する作用をもつことを示した。以上の結果から、この無血清培養法は神経生物学的研究において有用な分析系となりうることを指摘した

Alcoholic Liver Disease

Relationship between ethanol drinking and organs injury was reviewed and special emphasis was put on alcoholic liver disease. Consumption of alcoholic beverage expressed as ethanol per capita of adult in Japan increased 2.1 times in these 25 years and it is still increasing. Although the incidence of alcoholic liver disease in Japan also increased greatly during the above period, it seems likely that plateau level is coming because of genetically defined, unique type of alcohol metabolism in Japanese. Sex differences in susceptibility to alcohol were discussed. Among the six types of alcoholic liver disease, alcoholic liver fibrosis is relatively frequent in Japan. Mechanism of liver injury has been studied extensively. Alcohol itself is toxic but other factors such as dietary fat are also important. Biochemical and immunological markers of drinking were presented. As for the treatment, most patients especially in early stages of the disease well respond to alcohol withdrawal, but therapy of alcohol dependence in the background of the disease is very difficult requiring cooperative works of different kinds of specialists

Matsushiro Underground Cosmic-Ray Observatory (2S0 m. w. e. Depth) and the Observation of High Energy (r1012 /!V) Cosmic Ray Intensity Variation

A new underground cosmic-ray observatory was opened in Matsushiro, Nagano City, Japan on March 22, 1984, and a multi-directional muon telescope has been installed at an effective vertical depth of 220 m. w. e. underground. The telescope consists of 50 plastic scintillation detectors totally, arranged in two layers of 25 detectors each and has 17 directional channels of observation. We have made the continuous observation of the intensity variation of cosmic ray muons (median primary energies of detection d1012 eV) since that date. The intensity has been recorded every hour, and the average muon countingrates are; ～8.7×10t counts per hour for a wide-angle vertical telescope (two-fold coincidence between upper and lower arrays of detectors) and ～2.0×10t counts per hour for a vertical component-telescope, for example. In the present report, we describe briefly the underground observatory of Matsushiro and its surroundings, including the underground tunnel, the muon detector, the multi-directional telescope constructed and some of its related characteristics. We also present some of the observed intensity variations of cosmic ray muons for a full five-year period from April 1984 through March 1989 and discuss preliminarily the analyzed results of them in solar and sidereal time.Article信州大学理学部紀要 24(1): 1-46(1989)departmental bulletin pape

High mobility group box 1 complexed with heparin induced angiogenesis in a matrigel plug assay

Angiogenesis involves complex processes mediated by several factors and is associated with inflammation and wound healing. High mobility group box 1 (HMGB1) is released from necrotic cells as well as macrophages and plays proinflammatory roles. In the present study, we examined whether HMGB1 would exhibit angiogenic activity in a matrigel plug assay in mice. HMGB1 in combination with heparin strongly induced angiogenesis, whereas neither HMGB1 nor heparin alone showed such angiogenic activity. The heparin-dependent induction of angiogenesis by HMGB1 was accompanied by increases in the expression of tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor-A120 (VEGF-A120). It is likely that the dependence of the angiogenic activity of HMGB1 on heparin was due to the efficiency of the diffusion of the HMGB1-heparin complex from matrigel to the surrounding areas. VEGF-A165 possessing a heparin-binding domain showed a pattern of heparin-dependent angiogenic activity similar to that of HMGB1. The presence of heparin also inhibited the degradation of HMGB1 by plasmin in vitro. Taken together, these results suggested that HMGB1 in complex with heparin possesses remarkable angiogenic activity, probably through the induction of TNF-alpha and VEGF-A120.</p