Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2

不平表現方略の順序構造 : 日本人とタイ人英語学習者を対象にした比較研究

本研究の目的は，日本人・タイ人英語学習者の不平表現方略を調査・比較することである。「不平」とは，他者の過去や現在の行動に対する不満や不賛成を表現するものであり，深刻なフェイス侵害行為のひとつである。しかしながら，従来の研究では，依頼に代表される他の主要な発話行為と比較して，不平の発話行為はあまり研究の焦点となっていなかった。また，英語がグローバル言語として使用されているEnglish-as-a-Lingua-Franca 環境では，異なる母語や文化的背景を持つL2 学習者が，不平表現方略を同様に使用しているのか否かを検証することは重要であると考えられる。本研究では，日本人とタイ人の英語学習者を対象に，社会的地位（Power）と心理的距離（Distance）を統制した談話完成テスト（discourse completion test; DCT）を実施した。DCT は，アカデミックな文脈における4 つの状況（例：エッセイの成績について教授に不平を言う）で構成された。調査で得られた筆記データは，主要行為部（head act）と呼ばれる中核的な情報（例：私の評価が悪い理由を教えてください）と，補助手番部（supportive move）と呼ばれる周辺情報（例：こんにちは，お邪魔しますが，今，時間ありますか）に現れる言語的特徴の頻度の観点から分析された。さらに，両言語話者による不平表現方略を構成する上述の情報の出現順序についても分析された。その結果，タイ人学習者は不満をより直接的に表現するのに対し，日本人学習者は主要行為部を示す前に，より多くの補助手番部を提示する傾向があることが明らかになった。これらの結果は，母語や文化的背景の違いがL2発話行為の遂行に影響を与えることを示唆している。特に，本小論は異文化誤解に影響を与える可能性のある，方略の出現順序構造の特質を特定することに役立つと期待される。This study aimed to investigate complaint-making strategies used by Japanese and Thai learners of English. Complaining, an expression of dissatisfaction or disapproval toward others’ past or ongoing actions, is a highly face-threatening act. However, previous studies have paid less attention to this speech act as compared to other major speech acts, such as requests. Additionally, given that English is used as a global language, in an English-as-a-Lingua-Franca environment, it is important to examine whether L2 learners with different L1/cultural backgrounds use similar or different complaint-making strategies. In this study, Japanese and Thai university-level learners of English took a discourse completion test (DCT), which manipulated social status (power) and mental distance (distance). The DCT comprised four situations in academic contexts (e.g., complaining about an essay grade to a professor). Data were analyzed regarding the frequency of linguistic characteristics appearing in core information called head acts and surrounding information called supportive moves. Further, the sequential organization of explicit complaints in complaint-making strategies was also analyzed as a way of identifying culture-specific patterns of preference organization. Preliminary analysis indicated that Thai learners expressed complaints more directly, whereas Japanese learners tended to use more supportive moves before producing the head act. These results suggest that different L1/cultural backgrounds influence L2 speech act performance. The present study contributes to our understanding of the social and sequential organization of talk in cross-cultural interactions and its potential effects on intercultural miscommunication.This study was supported by KAKENHI Grant 17K02973 from the Japan Society for the Promotion of Science.This paper was originally read at the 2019 APLX International Conference on Applied Linguistics held at the National Taipei University of Technology (Taipei Tech) on October 31, 2019

Nasal delivery of Japanese cedar pollen Cryj1 by using self-gelling immunostimulatory DNA for effective induction of immune responses in mice.

To develop an immunotherapeutic vaccine for treatment of allergic rhinitis, we developed a controlled release formulation of Cryj1, a major Japanese cedar pollen allergen, with immunostimulatory potency. Two sets of hexapod-like structured DNA (hexapodna) were prepared using six oligodeoxynucleotides (ODNs) each, including ODNs with an unmethylated cytosine-phosphate-guanine (CpG) sequence (CpG motif), to obtain an immunostimulatory DNA hydrogel (sDNA hydrogel). A non-immunostimulatory DNA hydrogel (nsDNA hydrogel) was also prepared using ODNs with no CpG motifs. The sDNA hydrogel was more effective than its components or the nsDNA hydrogel for production of interleukin (IL)-12 after addition to murine macrophage-like RAW264.7 cells or after intranasal administration to mice. Then, a Cryj1-loaded sDNA hydrogel (Cryj1/sDNA hydrogel) formulation was prepared by mixing solutions containing both Cryj1 and hexapodna. Cryj1 was slowly released from the sDNA hydrogel in phosphate-buffed saline. After intranasal administration of the fluorescein isothiocyanate (FITC)-labeled Cryj1/sDNA hydrogel in mice, FITC-Cryj1 was retained in the nasal cavity for a longer period than FITC-Cryj1 mixed with hexapodna in solution. Intranasal immunization of mice with the Cryj1/sDNA hydrogel resulted in high levels of Cryj1-specific IgG in nasal lavage fluid (NFL), IL-12 and interferon-γ release from spleen cells after re-stimulation with Cryj1 when compared with intranasal immunization with the other formulations examined. These results indicate that the self-gelling immunostimulatory DNA hydrogel is an effective formulation for controlled induction of allergen-specific immune responses

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator

Injectable, self-gelling, biodegradable, and immunomodulatory DNA hydrogel for antigen delivery.

DNA nanotechnology-based nanosystems and macrosystems have attracted much attention in the biomedical research field. The nature of DNA endows these systems with biodegradable, biocompatible, and immunomodulatory properties. Here, we present an injectable hydrogel system that consists only of chemically synthesized short DNA strands, water, and salts. Several preparations of polypod-like structured DNA, or polypodna, were designed, including tri-, tetra-, penta- and hexapodna, as the building blocks of self-gelling DNA hydrogel. Under physiological conditions, properly designed polypodna preparations formed a hydrogel. The analysis of the modulus data of the hydrogel consisting of two sets of hexapodna preparations showed that this injectable hydrogel was reorganized at a time scale of 0.25s. Then, DNA hydrogel containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides was used to stimulate innate immunity through Toll-like receptor 9, the receptor for CpG DNA. Gel formation significantly increased the activity of immunostimulatory CpG DNA, retarded the clearance after intradermal injection into mice, and increased the immune responses to ovalbumin (OVA) incorporated into the hydrogel as a model antigen. OVA/CpG DNA hydrogel induced much less local or systemic adverse reactions than OVA injected with complete Freund's adjuvant or alum. GpC DNA hydrogel containing no CpG sequences was less effective, indicating the importance of immunomodulation by CpG DNA hydrogel. Thus, we have created an efficient system for sustained delivery of antigens or other bioactive compounds

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae.

<p>The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.</p