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

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

Desulfation of Heparan Sulfate by Sulf1 and Sulf2 Is Required for Corticospinal Tract Formation

Heparan sulfate (HS) has been implicated in a wide range of cell signaling. Here we report a novel mechanism in which extracellular removal of 6-O-sulfate groups from HS by the endosulfatases, Sulf1 and Sulf2, is essential for axon guidance during development. In Sulf1/2 double knockout (DKO) mice, the corticospinal tract (CST) was dorsally displaced on the midbrain surface. In utero electroporation of Sulf1/2 into radial glial cells along the third ventricle, where Sulf1/2 mRNAs are normally expressed, rescued the CST defects in the DKO mice. Proteomic analysis and functional testing identified Slit2 as the key molecule associated with the DKO phenotype. In the DKO brain, 6-O-sulfated HS was increased, leading to abnormal accumulation of Slit2 protein on the pial surface of the cerebral peduncle and hypothalamus, which caused dorsal repulsion of CST axons. Our findings indicate that postbiosynthetic desulfation of HS by Sulfs controls CST axon guidance through fine-tuning of Slit2 presentation

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+

E3 Ubiquitin Ligase Synoviolin Is Involved in Liver Fibrogenesis

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.The expression and localization of synoviolin in the liver were analyzed in CCl(4)-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno(+/-) mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno(+/-) mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno(-/-) mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno(-/-) MEF cells.In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno(+/-) mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno(+/-) mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis

Japanese Lung Cancer Society Guidelines for Stage IV NSCLC With EGFR Mutations

Patients with NSCLC in East Asia, including Japan, frequently contain EGFR mutations. In 2018, we published the latest full clinical practice guidelines on the basis of those provided by the Japanese Lung Cancer Society Guidelines Committee. The purpose of this study was to update those recommendations, especially for the treatment of metastatic or recurrent EGFR-mutated NSCLC. We conducted a literature search of systematic reviews of randomized controlled and nonrandomized trials published between 2018 and 2019 that multiple physicians had reviewed independently. On the basis of those studies and the advice from the Japanese Society of Lung Cancer Expert Panel, we developed updated guidelines according to the Grading of Recommendations, Assessment, Development, and Evaluation system. We also evaluated the benefits of overall and progression-free survival, end points, toxicities, and patients’ reported outcomes. For patients with NSCLC harboring EGFR-activating mutations, the use of EGFR tyrosine kinase inhibitors (EGFR TKIs), especially osimertinib, had the best recommendation as to first-line treatment. We also recommended the combination of EGFR TKI with other agents (platinum-based chemotherapy or antiangiogenic agents); however, it can lead to toxicity. In the presence of EGFR uncommon mutations, except for an exon 20 insertion, we also recommended the EGFR TKI treatment. However, we could not provide recommendations for the treatment of EGFR mutations with immune checkpoint inhibitors, including monotherapy, and its combination with cytotoxic chemotherapy, because of the limited evidence present in the literature. The 2020 Japanese Lung Cancer Society Guidelines can help community-based physicians to determine the most appropriate treatments and adequately provide medical care to their patients

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

A multi-ethnic meta-analysis identifies novel genes, including ACSL5, associated with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating progressive motor neuron disease that affects people of all ethnicities. Approximately 90% of ALS cases are sporadic and thought to have multifactorial pathogenesis. To understand the genetics of sporadic ALS, we conducted a genome-wide association study using 1,173 sporadic ALS cases and 8,925 controls in a Japanese population. A combined meta-analysis of our Japanese cohort with individuals of European ancestry revealed a significant association at the ACSL5 locus (top SNP p = 2.97 × 10−8). We validated the association with ACSL5 in a replication study with a Chinese population and an independent Japanese population (1941 ALS cases, 3821 controls; top SNP p = 1.82 × 10−4). In the combined meta-analysis, the intronic ACSL5 SNP rs3736947 showed the strongest association (p = 7.81 × 10−11). Using a gene-based analysis of the full multi-ethnic dataset, we uncovered additional genes significantly associated with ALS: ERGIC1, RAPGEF5, FNBP1, and ATXN3. These results advance our understanding of the genetic basis of sporadic ALS

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

BACKGROUND:Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. PRINCIPAL FINDINGS:Here, we demonstrate that CD4(+)CD25(+)CCR4(+) T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-gamma production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4(+)CD25(+)CCR4(+) T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-gamma-producing CD4(+)CD25(+)CCR4(+)Foxp3(-) T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. CONCLUSIONS:We have defined a unique T cell subset--IFN-gamma(+)CCR4(+)CD4(+)CD25(+) T cells--that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system