Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression

AbstractChronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB

惑星紫外放射の測定に向けた超小型観測器の開発

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 吉川 一朗, 東京大学教授 今村 剛, 東京大学准教授 井 通暁, 東京大学准教授 小泉 宏之, 東京大学准教授 笠原 慧, 東京大学講師 吉岡 和夫University of Tokyo(東京大学

Track-etched membrane-based dual-electrode coulometric detector for microbore/capillary high-performance liquid chromatography

The electrochemical flow cell containing track-etched microporous membrane electrodes was applied to a dual-electrode coulometric detector for microbore/capillary HPLC with a small injection volume and low eluent flow rate. The proposed flow cell with a 0.1-mm diameter inlet channel gave a detection volume of 0.08 nL per electrode, which was determined by the eluent flow through the electrode. For the dual-electrode detector, the calculated volume was 0.24 nL. The efficiency of electrooxidation of L-ascorbic acid increased as the flow rate decreased and was close to 100% when the flow rate was below 50 µL min−1, which is a common flow rate in microbore or capillary liquid chromatography. Catecholamines, such as noradrenaline, adrenaline, and dopamine, were detected by total conversion with two-electron oxidation in the potential range from 0.8 to 1.0 V vs. Ag/AgCl after separation with a microbore column. These peaks were accompanied by corresponding cathodic peaks derived from quasi-stable electrooxidation products of the catecholamines. The detection limits of noradrenaline, adrenaline, and dopamine were 0.1, 0.1, and 0.2 μM, respectively. The RSD values for five replicate measurements of 5.0 μM of these compounds were 0.9%, 0.7%, and 1.5%, respectively. Coulometric detection was also demonstrated by determination of catecholamines in pharmaceuticals

Evaluation of BACE1 Silencing in Cellular Models

Beta-secretase (BACE1) is the major enzyme participating in generation of toxic amyloid-beta (Aβ) peptides, identified in amyloid plaques of Alzheimer's disease (AD) brains. Its downregulation results in decreasing secretion of Aβ. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus) were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, βIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR), however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed

Initial Results for Science Instruments Onboard EQUULEUS During the Cruising Phase Toward the Earth Moon Lagrange Point

EQUULEUS (EQUilibriUm Lunar-Earth point 6U Spacecraft) is a spacecraft to explore the cis-lunar region including the Earth-Moon Lagrange point L2 (EML2). The spacecraft is being jointly developed by JAXA, the University of Tokyo, and several other universities in Japan. After being launched into a lunar transfer orbit by NASA\u27s SLS (Space Launch System) Artemis-1 on November 16, 2022, the spacecraft successfully performed a first Delta-V and a trajectory correction maneuver. This enabled a precise lunar flyby and successful insertion into the orbit toward EML2. Although the size of EQUULEUS is only 6U CubeSat, the spacecraft carries three different science instruments. The spacecraft can effectively demonstrate science missions during and after the flight to EML2 by using these instruments; the plasmasphere observation around the Earth by PHOENIX, the space dust flux detection in the cis-lunar region by CLOTH, and the lunar impact flash (LIF) observation at the far side of the moon by DELPHINUS. All instruments have already completed its checkout. During the cruising phase, PHOENIX conducted Earth observations and successfully identified the Earth\u27s plasmashere. CLOTH has started regular standby operations. DELPHINUS obtained impressive images such as the far side of the Moon at lunar closest approach and long-period comet, Comet ZTF. This poster presents the details of these scientific missions and the initial checkout and observation results of the science instruments

Spred2-deficiency enhances the proliferation of lung epithelial cells and alleviates pulmonary fibrosis induced by bleomycin

The mitogen-activated protein kinase (MAPK) pathways are involved in many cellular processes, including the development of fibrosis. Here, we examined the role of Sprouty-related EVH-1-domain-containing protein (Spred) 2, a negative regulator of the MAPK-ERK pathway, in the development of bleomycin (BLM)-induced pulmonary fibrosis (PF). Compared to WT mice, Spred2−/− mice developed milder PF with increased proliferation of bronchial epithelial cells. Spred2−/− lung epithelial cells or MLE-12 cells treated with spred2 siRNA proliferated faster than control cells in vitro. Spred2−/− and WT macrophages produced similar levels of TNFα and MCP-1 in response to BLM or lipopolysaccharide and myeloid cell-specific deletion of Spred2 in mice had no effect. Spred2−/− fibroblasts proliferated faster and produced similar levels of MCP-1 compared to WT fibroblasts. Spred2 mRNA was almost exclusively detected in bronchial epithelial cells of naïve WT mice and it accumulated in approximately 50% of cells with a characteristic of Clara cells, 14 days after BLM treatment. These results suggest that Spred2 is involved in the regulation of tissue repair after BLM-induced lung injury and increased proliferation of lung bronchial cells in Spred2−/− mice may contribute to faster tissue repair. Thus, Spred2 may present a new therapeutic target for the treatment of PF

Gluteal-fold adipofascial perforator flap transposition for rectourethral fistula reconstruction

If a rectourinary fistula does not close spontaneously, it requires surgical closure. We present our experience of rectourethral fistula reconstruction using a gluteal-fold perforator flap, resulting in a successful outcome. The patient was a 64-year-old man with prostate cancer who underwent radical prostatectomy. However, he developed rectourinary fistula, which required surgical closure. A dissection was undertaken to divide the fistula tract, and the rectal and urethral defect were closed. A 12.0×3.0 cm gluteal-fold adipofascial perforator flap was harvested and placed in the space between the rectum and urethra. The viability of lap was favourable, without infection or necrosis. The patient could walk the next day, and was discharged 2 weeks later without fecaluria or liquid stool. We conclude that the gluteal-fold adipofascial perforator flap offers excellent functional advantages in rectourethral fistula reconstruction with minimal morbidity at the donor site

Soft X-ray Absorption and Photoemission Studies of Ferromagnetic Mn-Implanted 3CC-SiC

We have performed x-ray photoemission spectroscopy (XPS), x-ray absorption spectroscopy (XAS), and resonant photoemission spectroscopy (RPES) measurements of Mn-implanted 3$C$-SiC (3$C$-SiC:Mn) and carbon-incorporated Mn$_{5}$Si$_{2}$ (Mn$_{5}$Si$_{2}$:C). The Mn 2$p$ core-level XPS and XAS spectra of 3$C$-SiC:Mn and Mn$_{5}$Si$_{2}$:C were similar to each other and showed "intermediate" behaviors between the localized and itinerant Mn 3$d$ states. The intensity at the Fermi level was found to be suppressed in 3$C$-SiC:Mn compared with Mn$_{5}$Si$_{2}$:C. These observations are consistent with the formation of Mn$_{5}$Si$_{2}$:C clusters in the 3$C$-SiC host, as observed in a recent transmission electron microscopy study.Comment: 4 pages, 3 figure