Prevotella intermedia のタンパク分解酵素の部分精製と性状

歯周病原菌の一つである偏性嫌気性グラム陰性桿菌Prevotella intermedia ATCC２５６１１株の培養上清中のタンパク分解酵素を硫酸アンモニウム塩析，イオン交換クロマトグラフィー，セファクリルゲル濾過で部分精製し，その酵素学的性状を調べた．タンパク分解活性はアゾ色素結合コラーゲン（アゾコル）を用いた．レマゾ－ルブリアントブルー結合ハイドパウダーの分解も見られたが，その活性はアゾコルに対して約５０％であった．また，フィブリン溶解とカゼイン分解活性も認められた．分子量はゲル濾過法で２８kDa と算定され，本酵素はセリン酵素阻害剤と金属キレーターで強く阻害されたが，システイン酵素の阻害剤および還元剤による影響はなかったので，メタロ・セリン酵素に分類される．反応の至適pH は７．０～７．５にあり，酵素の５０％失活に要する時間は，５０℃加熱で２５分を要したが，６０℃では５分であった．P. intermedia のペプチダーゼについての報告はかなりなされているが，無論ぺプチダーゼはタンパク質に直接作用するのではなく，ペプチダーゼが働くにはタンパク質からのペプチドの蓄積が必要である．しかし，それを供給する本菌のタンパク分解酵素についての知見はまだ不十分であり，さらに詳しい性状把握ががなされるべきである．我々も今後，このレポートで扱ったタンパク分解酵素の，完全精製と合成基質の探索をし，より詳細な研究を推進する必要があると考える

Porphyromonas gingivalis ４株における病原性関連酵素とタンパクの比較研究

Porphyromonas gingivalis の研究上使われる代表的な４株（ATCC３３２７７，３８１，W５０，W８３）を用いて，病原因子と深い関係のある，RGP，KGP を含むプロテイナーゼ，ペプチダーゼおよびヘモグロビンとミオグロビン結合タンパクを通しての結合活性について比較検討した．プロテイナーゼに関して，４株ともにその産生が見られ，それぞれの株で３画分での分布状況を調べたところ，RGP，KGP，アゾコル分解酵素ともに，培養上清，粗抽出液，エンベロープの順に多かった．RGP，KGP に関しては，ATCC３３２７７，３８１，W５０で培養上清にほぼ６０％ほど含まれていたが，W８３では９４％を含んでいた．これに反して，ペプチダーゼ（PTP，DPP−I，DPP−II，DPP−IV）は４株ともに培養上清には検出されず，そのほとんどが粗抽出液に認められ，数％のみがエンベロープに分布しており，ペプチダーゼには細胞外への分泌機構が作用しないものと思われる．いずれの株でもPTP 産生単位数がDPP のどれよりも高かった．菌体細胞のヘムタンパクへの結合もいずれの株でも見られたが，ともにpH 依存性が見られ，酸性で強く，中性，アルカリでは非常に弱かった．結合の度合いはヘモグロビンとミオグロビンでは違いはほとんど認められず，これはミオグロビンがヘモグロビン分子の四分の一から成っていることから当然と言えよう．他のヘムタンパク（チトクロームC，カタラーゼ）への結合も弱いながら観察された

Streptococcus anginosus のプロリルトリペプチジルペプチダーゼの産生と酵素性状

Streptococcus anginosus is considered to be implicated in the etiology of oral infectious diseases as well as abscess formation in various body sites. We investigated the production and the enzymatic properties of PTP of S. anginosus NCTC 10713. This enzyme was found only in cell extract and active on tripeptide substrates containing proline residue at P1 position, particularly H−Ala−Ala−Pro−p−nitroanilide. The enzyme was produced by all 8 species of tested streptococci, indicating occurrence of this enzyme is rather ubiquitous within streptococci. This PTP was purified to homogeneity from the cell extract by the procedures including ammonium sulfate precipitation, chromatography, gel filtration and electrophoresis. The enzyme was inhibited by serine enzyme inhibitors and chelating reagents, indicating this PTP is a serine metalloenzyme with a molecular mass of 66 kDa. The enzyme was active against H−Ala−Ala−Pro−p−nitroanilide and H−Ala−Phe−Pro−p−nitroanilide in neutral pH solutions. The activity was completely lost by heating at 50°C for 10min

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis

Porphyromonas gingivalis のプロリルトリペプチジルペプチダーゼの産生，単離および性状

Formation, cellular locations, isolation and enzymatic properties of PTP of Porphyromonasgingivalis, an anaerobic periodontal pathogen were investigated. Almost all activities of this enzyme were detected in the de extract of the cell, but the other bacterial fractions such as culture fluids, envelopes and vesicles were not found to contain PTP. PTP was purified from the crude extract prepared by sonication and centrifugation through five steps including concentration, ion exchange hromatography, gel filtration, hydrophobic interaction chromatography and isoelectric focusing to homogeneity. The enzyme was a serine enzyme since it was inhibited strongly by Pefabloc SC, propylfluorophosphate and 3,4–dichloroisocoumarin. It hydrolyzed H–Ala–Ala–Pro–pNA and H–Ala–Phe–Pro–pNA. The molecular mass was determined as 45 kDa and isoelectric point was 5.₇. Optimum pH was moderately broad, and maximum activity was observed in the range of pH ₇.0 to 9.0. The residual activity after heating at 50℃ for 5 min was 29%, but heating at 60℃ resultedin complete loss of the activity.松本歯科大学大学院歯学独立研究科博士（歯学）学位申請論文 ; 大学院歯学独立研究科　硬組織疾患病態解析学（主指導教員：長谷川博雅

Prevotella intermedia の細胞外タンパク分解酵素の補充的研究：産生と酵素性状

Formation and some enzymatic properties of a proteinase of Prevotella intermedia ATCC 2₅611 were examined using Azocoll, a solid chromogenic non–specific proteinase substrate. Anaerobic globe box supported better growth of cell and production of proteinase than AnaeroPak. Nearly all proteinase activity was found in the extracellular culture fluid, but not in the cell extract and the envelope. Production of proteinase started immediately after inoculation of the bacteria to the medium and continued until the middle stage of the stationary phase of the growth. Proteinase production was strongly inhibited by addition of glucose or fructose to the medium, moderately by sucrose and weakly by galactose. This proteinase was inactivated entirely by heating at 60℃ for 20 min. Ethyleneglycol bis (β –aminoethyl ether)–N,N,N,N ʼ–tetraacetate (EGTA), Zn2+ and Cu2+ completely inhibited the proteolysis. No effect on the activity was observed by Antipain, SDS, N–tosyl–L–phenylalanyl oromethyl ketone (TPCK), Ca2+ and Mg2+

Porphyromonas gingivalis のジペプチジル ペプチダーゼ–IV の性状

Dipeptidyl peptidase IV (DPP–IV) was purified to homogeneity from the crude extract of Porphyromonas gingivalis. Isoelectric point and molecular weight of DPP–IV were 6.1 and 66 kDa, respectively. Km value for the appropriate substrate (H–Gly–Pro–pNA) was 0.2₅ mM, and Vmax/Km was 0.₅2. Optimum pH for the activity was found at pH ₇.₅. DPP–IV was considered a serine enzyme

Sophocles, <i>Aleadae</i>: a Reconstruction

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Notes on Sophocles’ <i>Epigoni</i>

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The relationship between a low grain intake dietary pattern and impulsive behaviors in middle-aged Japanese people

Several studies indicate that dietary habits are associated with mental health. We are interested in identifying not a specific single nutrient/food group but the population preferring specific food combinations that can be related to mental health. Very few studies have examined relationships between dietary patterns and multifaceted mental states using cluster analysis. The purpose of this study was to investigate population-level dietary patterns associated with mental state using cluster analysis. We focused on depressive state, sleep quality, subjective well-being, and impulsive behaviors using rating scales. Two hundred and seventy-nine Japanese middle-aged people participated in the present study. Dietary pattern was estimated using a brief self-administered diet-history questionnaire (the BDHQ). We conducted K-means cluster analysis using thirteen BDHQ food groups: milk, meat, fish, egg, pulses, potatoes, green and yellow vegetables, other vegetables, mushrooms, seaweed, sweets, fruits, and grain. We identified three clusters characterized as "vegetable and fruit dominant," "grain dominant," and "low grain tendency" subgroups. The vegetable and fruit dominant group showed increases in several aspects of subjective wellbeing demonstrated by the SF-8. Differences in mean subject characteristics across clusters were tested using ANOVA. The low frequency intake of grain group showed higher impulsive behavior, demonstrated by BIS-11 deliberation and sum scores. The present study demonstrated that traditional Japanese dietary patterns, such as eating rice, can help with beneficial changes in mental health