High Throughput Screening and mRNA-Display Selection for the Identification of Biologically Functional Molecules

Biologically functional molecules with desired properties have proven to be invaluable tools for investigating biological systems. Moreover, the identification of those functional molecules is a crucial task in modern drug discovery. These goals can be achieved by two major approaches: gscreeningh and gselectionh. Chapter 2 introduces one attractive method for gscreeningh to identify small molecule inhibitors of a given protein target from a small organic chemical library. The reverse transcriptase (RT) plays a crucial role in the early steps of the life cycle of HIV. Therefore HIV-1 RT serves as one of the major drug targets in anti-HIV therapy. Although current therapy for HIV-infected patients involving a combination of three or more of the following drugs: RT inhibitors, protease inhibitors, and/or viral entry inhibitors, has been highly successful, its long-term efficacy is severely limited by the emergence of drug-resistant variants of HIV. One way to address this problem is to identify new type of drugs with different inhibitory mechanisms to combat HIV. Thus, HIV-1 RT served a valuable target in the herein depicted screening approach. Protein-dependent alosteric ribozymes (or reporter ribozymes) are powerful tools which are able to sense molecular interactions, e.g. protein-protein, protein-nucleic acid, or protein-metabolite interactions in real-time. The expedience of this assay format makes it applicable to and suitable for high throughput screening in order to search for new molecules which disrupt such interactions. Reporter ribozyme FK-1 is a rationally engineered hammerhead ribozyme in a fusion construct with anti-RT aptamer, which can detect the presence of HIV-1 RT selectively in a domain specific manner. Thus, FK-1 enables to identify compounds via FRET-based fluorescence readout, which compete for the binding to HIV-1 RT with aptamer sequences inserted in the ribozyme construct. Through screening of 2500 small molecules using the reporter ribozyme (FK-1)-based assay, three potential inhibitors (28F6, 3E4 and 2E10) of HIV-1 RT were identified with in vitro inhibitory concentration that gave half-maximal activity (IC50) of 2-5 µM on DNA-dependent DNA polymerase activity. The three compounds were re-synthesized and further investigated to evaluate their selectivity and to elucidate the inhibitory mechanism. The selectivity was determined by testing inhibitory effect of the compound on other RTs (AMV and MMLV) and DNA polymerases from both prokaryotic (Klenow Fragment) and eukaryotic (human DNA polymerase ƒÀ) sources. Among the three compounds, 3E4 showed not only the lowest IC50 value for HIV-1 RT (2.1 µM) but the highest discrimination from all the other RT and DNA polymerases (10~50 fold) in terms of DNA-dependent DNA polymerase activity. The selectivity of 3E4 with the cognate RT from HIV-2 was a factor of approximately 4.5. In addition, the interaction between 3E4 and HIV-1 RT was observed by a surface plasmon resonance-based biosensor. Moreover, a cell-based assay for phenotypic analysis of sensitivity of HIV-1 to the three inhibitors using a self-inactivating virus vector system demonstrated that 3E4 inhibited marker gene expression in concentration dependent manner with a IC50 value of 5.3 µM. The effect of 3E4 was further confirmed with the similar reduction of HIV-1 replication and infectivity in vivo using a HIV wild type strain. In summary, the data strongly indicates that the small molecule 3E4 identified by reporter ribozyme-based screening is a highly specific and cellular active HIV-1 RT inhibitor, which has significant potentiality as novel type of anti-HIV drug. Furthermore, the approach using a reporter ribozyme assay could be broadly applicable as screening format. In chapter 3, the challenge of a gselectionh approach has been performed with the aim to evolve functional proteins from a random sequence protein library. Prior to the selection, the protein library consisting of a random 88 amino acid sequence was constructed by short cassettes ligations to avoid stop codons and frameshift mutations. The design of secondary structural patterns like ƒ¿-helix and ƒÀ-strand was introduced into the random regions. A display technology called mRNA-display was exploited to prepare protein library, where each protein sequence was covalently linked through its carboxy terminus to the 3f end of its encoding mRNA. Starting with a library that contained 1 ~ 1011 - 5 ~ 1012 different proteins, three in vitro selections were attempted to identify protein binders to small molecules such as cibacron blue 3GA and coenzymes (NADP and CoA). The selection against cibacron blue 3GA yielded enriched fractions retaining on the column matrix after the ninth cycle. These sequences could not be eluted by NAD, the structural analogue of cibacron blue 3GA, even at higher concentration. The small fractions eluted with NAD from this column have been cloned and sequenced, resulting in unexpectedly high frequency of frameshifts and internal stop codons. The alignment of full length intact sequences exhibited no conserved motif but the most appearance of ƒ¿ƒÀƒ¿ƒÀ type of cassette. The remaining column fractions were able to be eluted with free cibacron blue 3GA, implying those sequences might be specific cibacron blue binders. Other selections against both NADP and CoA did not enrich protein binders. The observed problems through the experiment concerning poor solubility and fold instability of mRNA-displayed proteins should be improved for further investigations

同志社女子大学看護学部におけるキャリア教育の現状と今後の展望について

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Tamoxifen-induced ovarian hyperstimulation during premenopausal hormonal therapy for breast cancer in Japanese women

Purpose: Tamoxifen is an anti-estrogenic drug that is widely used for endocrine-dependent breast cancer as adjuvant hormonal therapy, and its use has been reported to be frequently associated with high levels of serum estradiol. Since the population of premenopausal women receiving tamoxifen therapy is growing in Japan, we retrospectively analyzed the incidence of ovarian hyperstimulation by tamoxifen therapy in Japanese women. Methods: Eleven patients who received surgical therapy for endocrine-dependent breast cancer and showed high values of serum estradiol during post-operative tamoxifen therapy were recruited in this study and evaluated by examining the serum concentration of follicular stimulating hormone (FSH) and follicular development. Results: The mean age, serum concentrations of estradiol and FSH, and follicular diameter were 41.3 years old, 1015.8 pg/mL, 11.8 mIU/mL, and 3.47 cm, respectively. In 6 cases, multiple follicular development was observed, while the other cases showed single follicular development with a mean serum estradiol level of 848.6 pg/mL and follicular diameter of 4.46 cm. There was no significant difference in age or FSH concentration between the two groups. The mean periods from the start of the single administration of tamoxifen to the initial detection of a high estradiol concentration was 716.5 days. Conclusions: These findings indicate that tamoxifen could stimulate the ovarian function even after 2-year treatment. Since single and multiple follicular developments with large sizes were observed, dual mechanisms through the inhibition of both negative and positive feedback to the hypothalamic-pituitary-axis can be proposed to explain the adverse effects of tamoxifen on ovarian function. © 2015, Yamazaki et al

Paired Activating and Inhibitory Immunoglobulin-like Receptors, MAIR-I and MAIR-II, Regulate Mast Cell and Macrophage Activation

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses

病名における「-性」の分析 : 一般書籍との比較から

Osaka UniversityOsaka UniversitySeinan Jo Gakuin UniversitySeinan Jo Gakuin UniversityNational Institute for Japanese Language and Linguistics会議名: 言語資源活用ワークショップ2020, 開催地: オンライン, 会期: 2020年9月8日−9日, 主催: 国立国語研究所 コーパス開発センター医療用語（病名）には，「-性」という語構成要素を含む合成語が多いが，どのような要素と「性」が結合するのか，また，どのように病名を構成するのかについては未だ詳らかではないといえる。本発表では，実践医療用語辞書ComeJisyo の見出し語を対象に「-性」を含む病名を調査し，その特徴について分析した。また，BCCWJ の書籍サブコーパスにおける「-性」の用例と比較することで，得られた特徴が病名特有のものであるかを検討した。病名における「-性」では「先天性」「多発性」「急性」などが高頻度に用いられ，「急性細菌性髄膜炎」のように他の「-性」との共起も見られる一方，書籍では「-性」の連続は見られず，「可能性」「必要性」のようにそれ自体が主語となる（体言用法の）ものが高頻度であるという違いが見られた。病名においては，一語で的確に症状を表現する必要があるため，「-性」を用いた細分化が行われていると考えられる

病名を表す合成語の語末調査

Seinan Jo Gakuin UniversitySeinan Jo Gakuin UniversityOsaka UniversityOsaka UniversityNational Institute for Japanese Language and Linguistics会議名: 言語資源活用ワークショップ2020, 開催地: オンライン, 会期: 2020年9月8日−9日, 主催: 国立国語研究所 コーパス開発センター本研究では合成語の語末により病名の判別が可能か否かを確認するために合成語の語末調査を行った。具体的には、病名を表す合成語5,465 語について語末のunigram、bigram、trigram を調べた。加えて、筆者等が着手している電子カルテに記載された合成語を対象とした語構成要素解析で定めた語単位で、合成語を分割した場合に語末となる語構成要素の頻度を調べた。その結果、（1）右側主要部の規則による意味を用いた判別が可能なこと、（2）病名の末尾には「ヘモクロマトーシス」のようなカタカナ語があり、文字単位での判別より語単位の判別の方が適していることが分かった。また、意味ラベル「接尾語」が付与された語構成要素と、「病名」が付与された語構成要素の内「接尾語」を含まない要素を用いることで「病名」の機械的な判別が可能であることが示唆された

「急性」を含む病名の語構成

Seinan Jo Gakuin UniversitySeinan Jo Gakuin University, PhysicianOsaka UniversityPhysicianNational Institute for Japanese Language and Linguistics会議名: 言語資源ワークショップ2022, 開催地: オンライン, 会期: 2022年8月30日-31日, 主催: 国立国語研究所 言語資源開発センター「急性骨髄性白血病」のように「～性」が複数含まれ、かつ「急性」を含む病名において、「急性」の緊急度と語順の関係を調べるために、『現代日本語書き言葉均衡コーパス』(BCCWJ)に出現した当該の病名28語について、BCCWJ、医師経過記録、多職種共有の経過記録での出現頻度を求めた。その結果、①「急性」は語頭に多く出現すること、②医療現場では使われない病名があること、③医療現場では「急性」無しの病名が多く使われていること、④「急性」とその他の「～性」の語順を変えた同義語が存在することが分かった

実践医療用語 語構成要素語彙試案表 Ver.2.0 の構築

Osaka UniversityKyorin UniversitySeinan Jo Gakuin UniversitySeinan Jo Gakuin University, PhysicianPhysicianKansai UniversityNational Institute for Japanese Language and Linguistics会議名: 言語資源ワークショップ2022, 開催地: オンライン, 会期: 2022年8月30日-31日, 主催: 国立国語研究所 言語資源開発センター医療記録データには、複数の単語が連結された合成語が多く存在する。そのため、自然言語処理を効率的に行うためには、合成語の語構成や、それらの構成要素の意味に着目し、合成語の構造を明らかにする必要がある。しかし、医療記録は非公開という資料的特質のため、言語学的な調査があまり行われてこなかった。また、医療関係者における意味のある言語単位も定まっておらず、整理の必要があった。こうした背景に基づいて作成した言語資源が『実践医療用語_語構成要素語彙試案表 Ver.2.0』である。本試案表は、『実践医療用語辞書ComeJisyoSjis-1』より抽出した合成語より作成した『実践医療用語_語構成要素語彙試案表Ver.1.0』を更新したもので、7,087語の合成語について、それぞれを構成する語構成要素6,633種と、語構成要素に付与した意味ラベル41種を収録している。本発表では、Ver1.0からの変更点と、本言語資源の特徴、意味ラベルに注目した語構成要素について概観を行った

High-resolution seismic reflection profiling across the Shiraiwa fault, eastern margin of the Yokote basin fault zone, northeast Japan : data acquisition and processing

The eastern margin of the Yokote basin fault zone extends about 56km at the western foot of the Ou Backbone Range, northeast Japan. The Rikuu earthquake (M=7.2) occurred in the Ou Backbone Range (Mahiru Range) on 31st August, 1896. Associated with this earthquake, four thrust faults-Obonai, Shiraiwa, Ota, and Senya fault3 appeared on the surface of the western foot of the Mahiru Range. These faults were highly sinuous with numerous gaps and en echelon steps. We conducted a high-resolution seismic reflection profiling survey across the Shiraiwa fault. The obtained seismic reflection data were processed by conventional common mid-point methods, post-stack migration, and depth conversion. The subsurface structure across the Shraiwa fault is characterized by branched low-angle reverse faults and conjugate back-thrust. The emergent thrust associated with the 1896 earthquake is regarded to be a subsidiary reverse fault