大腸菌を用いたフォスファカン（コンドロイチン硫酸プロテオグリカン）の融合コア蛋白の発現条件の検討

Optimal conditions for expressing a specific region of core protein of phosphacan, a chondroitin sulfate proteoglycan known as receptor type protein tyrosine phosphatase, as fusion protein with glutathione S-transferase (GST) in E.coli were examined. DNA fragments inserted into the expression vector (pGEX-4T-1) were amplified by RT-PCR using mRNA purified from E18 rat brain as template. Primers attached with BamH I or EcoR I restriction site on 5' end were used to amplify first strand cDNA by PCR. Before ligation into the pGEX-4T-1 for GST fusion protein, PCR products were once cloned using T-A cloning system because they were not directly ligated into the pGEX-4T-1. E.coli strain BL21 was transformed by pGEX-4T-1 ligated with restriction DNA fragment cut out from pCR II plasmid vector of T-A clonig system. The growth of transformed BL21 was not different between the colony incubated at 37℃ for 24-48h and the colony stored at 4℃ for 7-10 days after 24h incubation at 37℃. The desirable OD(550) of culture medium for inducing the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG) was from 0.6 to 1.0, because expression of native E.coli proteins per ml of culture medium was increased relatively when IPTG was added at OD(550) more than 1.0. The expression of fusion protein reached plateau around 6h after the induction. Relative expression of native E.coli proteins per ml of culture medium increased thereafter. Therefore, it may be desirable to purify the fusion protein around 6h after the induction.コンドロイチン硫酸プロテオグリカンの一種であるフォスフォアカンのコア蛋白の特異領域を、グルタチオン-S-トランスフェラーゼ（GST）との融合蛋白として大腸菌内で発現させ、その発現条件の検討を行った。胎生18日目のラット脳から抽出したmRNAを鋳型として、RT－PCRによって増幅したDNAフラグメントを発現ベクターに挿入した。PCRの為のプライマーは、BamH IとEcoR Iの酵素消化部位を5'末端に組み込んだものを用いた。GST融合蛋白を作製するために、PCR産物を一旦、T-Aクローニングシステムに組込み、その後プラスミド精製と制限酵素消化によって目的のフラグメントを切り出した後、pGEXベクターに再度組み込ませ、大腸菌（BL21）を形質転換した。形質転換した大腸菌（BL21）について、24～48時間培養後に、37℃で保存したものと、37℃で24時間培養後に4℃で7～10日間保存したものとの増殖曲線を比較したところ、両者に有意な差は認められなかった。融合蛋白の誘導に必要なイソプロピルチオ－β－D－ガラクトシド（IPTG）の添加の時期は、菌培養液の吸光度（550nm）が1.0以上の場合には融合蛋白に比べて他の大腸菌固有の蛋白の割合が相対的に増大してしまう為、また、吸光度が0.6以下では菌量が少ない為、吸光度が0.6～1.0を示す時期に添加する方がより望ましいことがわかった。IPTGによる誘導後、融合蛋白の発現は6時間でプラトーに達し、その後は大腸菌固有の内在蛋白量の割合が相対的に増加した。以上の結果より、融合蛋白の発現を誘導するための至適条件は次のように決定された。1. IPTG誘導は、培養液の吸光度（550nm）が0.6～1.0の際に開始する。2. IPTGによる誘導時間は、6時間とする

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

変形性膝関節炎患者膝関節液中の細胞外マトリックス成分と可溶性VCAM-1

It is generally accepted that the number of patients with osteoarthritis (OA) of the knee must increase in the future because of the increase of the aged. The pathogenic mechanisms of OA haven't been elucidated yet, but it is supposed that mediate pannus may cause the factors for irreversible cartilage damage in the inflammatory process. Therefore it is very important to find and predict OA at the early stage. Now there were many indices to determine joint damage with OA, for example, cytokines including IL, TNF, TGF, matrix metalloproteinase (MMPs), tissue inhibitor of metalloproteinase (TIMPs), proteoglycan (PG), hyaluronate (HA), super oxide, adhesion molecules including, ICAM-1, VCAM-1 and PECAM. In this study, we measured that the soluble VCAM-1 (sVCAM-1), soluble collagen (sCOLL), heparansulfate proteoglycan (HSPG), uronic acis and CRP in synovial fluids (SF) with OA. A positive correlation between the level of sVCAM-1 and sCOLL (ρ = 0.652) was noted. However there was no factor showing a correlation with CRP. It was suggested that the levels of sVCAM-1 and sCOLL in the synovia fluid were active inflammation indices of knee damage, as the different aspects from CRP. The level of HSPG also showed a unique trend because HSPG on the cell surface was quickly digested endocytosis, so that it was not suitable for the inflammatory index in synovial fluid with OA.変形性膝関節炎(OA)患者は,高齢化社会の到来とともに今後ますます増加するものと考えられる｡OAの発症メカニズムについては,関節軟骨の破壊･修復やパンヌス形成に関わる多くの関連因子(サイトカイン,マトリックス分解酵素,分解酵素抑制因子,プロテオグリカン,活性酸素,細胞接着因子など)について,多面的に研究が進んでいる｡しかし,膝関節の炎症初期の病態のメカニズムについては,なお詳細に解明されておらず,関節炎初期の炎症指標の検索が今後さらに必要と考えられる｡本研究で私たちは, OA患者膝関節液中の細胞外マトリックス構成成分であるコラーゲン,へパラン硫酸プロテオグリカン,ウロン酸および炎症の最も初期に血管内皮細胞に出現するVCAM-1等の可溶化成分濃度を測定した｡患者膝関節液(SF)中の可溶性コラーゲン量と可溶性VCAM-1(sVCAM-1)量とは有意な正の相関関係が(ρ=0.652)観察された｡しかし,ヘパラン硫酸プロテオグリカン(HSPG)と可溶性コラーゲン(sCOLL)値間には有意な相関関係は確認されなかった｡また,一般に炎症の指標として用いられるCRP値とも比戟検討したが,CRP値と他の測定項目値とは一定の相関関係は存在しなかった。以上の結果より,OA患者のSF中の炎症マーカ-として,急性炎症時には従来の炎症指標蛋白質であるCRP値が有力であるというSipe JDの報告から推測するとsCOLLとsVCAM-1はCRP値と一定の相関を示さないことより,CRPの動態とは異なった炎症指標としての有用性が考えられる｡しかし,HSPGはエンドサイトシスにより速やかに代謝されるためSF中への解離が少なく,炎症指標としての可能性は低いと考えられる

The Neurophysiological Implications of an Atypical Slow Negative Potential in Short Interval CNV Paradigm

We recorded a slow negative potential from Cz (10/20 method) in 49 healthy students (12 male, 37 female, mean age 19.1) by a short interstumulus interval CNV paradigm. The interstimulus interval was 2 or 3 seconds, the warning stimulus presented at random or regular interval at 0.2 Hz. An atypical negative variation with two separated negative peaks was recorded in 26.0-30.6% trials regardless of interstimulus interval or modality of warning stimulus presentation, while a typical CNV was recorded in 32.0-59.2% of trials. No apparent negative variation was recorded in 14.3-18.4% in 2 seconds interstimulus interval, and 28-38% in 3 seconds interstimulus interval, showing that 2 seconds interval is better to get stable CNV recording than 3 seconds interval. The first negative wave of the atypical negative variation was 692-799msec in duration, but frequently prolonged to 1000msec or more in 3 seconds interval. It usually had negative peak around 900-1100msec, but sometimes around 1500msec. This features are different from any reported negative components of CNV. The second negative wave began 800-1200msec before second stimulus, and had its peak just before second stimulus, showing common features with readiness potential. The appearance of CNV was unsatble in the students in which the atypical negative variation was recorded in regular, 2 seconds intersitimulus interval, and the amplitude of slow vertex response and pattern reversal visual evoked potential was lower in thses students than in the students in which a typical CNV was recorded more than 3 times in total 4 times of trials. These findings indicate that the atypical variation observed in this study is due to a lowered arousal level or cortical neuronal activity, rather than a separated appearance of different components of CNV.CNVの記録条件を検討する目的で、健康若年成人49名（男12、女37）を対象にS(1)-S(2)+Rパラダイムを用いてCNVを記録した。S(1)-S(2)間隔2秒のほうが3秒よりも安定してCNVが記録されたが、刺激間隔とは無関係に26～30％の試行で二峰性の陰性変動をもつ非定型な緩電位が記録された。この緩電位は同一被検者でも出現性が一貫せず、個体差よりも被検者の状態に依存していた。二峰性陰性波のうち、後期成分は準備電位に対応するものと思われたが、前期成分はS(1)後900～1100msecにピークをもつ陰性波で、これまで報告されたCNVの要素波とは対応しなかった。また、この非定型CNVを示す被検者ではCNV出現が不安定で、SVRおよびパターンVEPの振幅が低い傾向があり、試行時の被検者の覚醒水準低下よるものではないかと考えられた

An experimental study on the relation of T2-signal high intensity in MRI to histopathological changes in the kainic acid model of temporal lobe epilepsy in rats.

側頭葉てんかんでは，てんかん焦点に一致してMRI T2高信号領域が見られ，FLAIR法でこれがより明瞭になるが，このMRI所見と病理組織学的変化との関係は必ずしもはっきりしていない。そこで，Sprague－Dawleyラットにカイニン酸（KA）でけいれん発作重積状態を起こし，経時的にMRIを記録するとともに，ニッスル染色，GFAP免疫染色での病理組織学的変化を調べて両者の関係について検討した。KA群では，MRIで1～8週間後のいずれにおいてもpiriform cortexからentorhinal cortexにかけて不整形のT2高信号領域がみられたが，stage3のけいれん発作しか出現しなかったラットではstage4，5が出現したラットに比べて程度が弱かった。組織学的には，CA1，subiculum，piriform cortex，entorhinal cortexで神経細胞の消失，濃染細胞の増加と萎縮，GFAP免疫反応の増強が見られたが，piriform cortex，entorhinal cortexでの神経細胞消失の程度はT2信 号の程度と相関せず，GFAP免疫反応が増強した領域に一致して高信号がみられた。しかし，海馬のGFAP免疫反応増強はMRI所見に反映されず，これはMRIの解像度の限界にもよると考えられた。The relation of T2-signal high intensity areas observed in temporal lobe epilepsy to histopathological changes in limbic structures was examined in the rat kainic acid (KA) model of epilepsy. Male 8-week-old Sprague-Dawley rats were injected with 10mg/kg(i.p.) of KA or saline (control). Repetitive generalized convulsions (stage 3, or stage 4,5 seizures of amygdala kindled seizures) lasted for 3 to 4 hrs. were induced by KA injection in all rats. MRI was recorded on the day before the KA injection and 1, 2, 4, 8 weeks after the injection by fluid-attenuated inversion recovery method under deep pentobarbital anesthesia. Following the last MRI recording, rats were perfused with 4% paraformaldehyde (PFA) from left cardiac ventricle, post-fixed overnight in 4% PFA and brains were embedded in parafin. Coronal brain sections (6μm) were stained with cresyl violet, or mouse anti-GFAP antibody followed by biotinylated goat anti-mouse IgG and avidin-biotin-peroxidase (vectastain ABC kit). Irregularshaped moderate to severe high T2-signal areas were observed in bilateral piriform and entorhinal cortex in MRI. These high T2-signal areas were ovserved from 1 week after the KA injection to 8 weeks after the injection, and were more prominent in rats elicited stage 4 or 5 seizures than in rats elicited stage 3 seizures. Loss of pyramidal neurons and increased GFAP immunoreactivity were observed in piriform cortex, entorhinal cortex, CA1, subiculum, and hilus of dentate gyrus. The increase of GFAP immunoreactivity, but not the intensity of neuronal loss, in piriform and entorhinal cortex was almost correponded to the size and intensity of T2-signal. However, the increase of GFAP immunoreactivity in hippocampus was not detected as the increase of T2-signal in MRI. These findings indicate that astroglial reactions in piriform and entorhinal cortex are more sensitive to T2-weighted MRI than those in hippocampus

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

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

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

公的統計ミクロデータ利用の新時代 －情報・システム研究機構による利用支援－

ISM Online Open House, 2021.6.18統計数理研究所オープンハウス（オンライン開催）、R3.6.18ポスター発

統計数理研究所におけるオンサイト分析拠点形成

Open House, ISM in Tachikawa, 2013.6.14統計数理研究所オープンハウス（立川）、H25.6.14ポスター発