All-or-none switching of transcriptional activity on single DNA molecules caused by a discrete conformational transition

Recently, it has been confirmed that long duplex DNA molecules with sizes larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an elongated coil state to a compact globule state upon the addition of various kinds of chemical species that usually induce DNA condensation. In this study, we performed a single-molecule observation on a large DNA, Lambda ZAP II DNA (ca. 41 kbp), in a solution containing RNA polymerase and substrates along with spermine, a tetravalent cation, at different concentrations, by use of fluorescence staining of both DNA and RNA. We found that transcription, or RNA production, is completely inhibited in the compact state, but is actively performed in the unfolded coil state. Such an all-or-none effect on transcriptional activity induced by the discrete conformational transition of single DNA molecules is discussed in relation to the mechanism of the regulation of large-scale genetic activity.Comment: 14 pages, 2 figure

Studies on the mechanism of phagocytosis. I. Effect of metabolic inhibitors on the phagocytosis of iron colloid particles by ascites macrophages

For the purpose to clarify the mechanism of phagocytosis or pinocytosis, the observations on the tumor ascites, including the macrophages as well as the tumor cells, were carried out by incubating with the iron colloid with or without pretreatment by several inhibibitors of glycolysis, oxidative phosphorylation and respiration, or under hypotonic or cold environments. The results have demonstrated that there are three steps in the phagocytosis. The first step is the adhesion of the substance to the cell surface, which is not an energy-requiring process. The second step is the engulfing which proceeds by using the energy supplied by glycolysis. The third is the accumulation of the substance into the vesicles through the canaliculi connecting the cell surface with the vesicles. The discussion was made on the existence of the active site on the cell surface to which the substance can be adhered, and the accumulation mechanism of the material into the phagocytic vesicles by the membrane flow, the flowing movement of the outer lipid layer of a unit membrane through the canaliculi which connect the cell surface to the phagocytic vesicles.</p

半導体光触媒の構造的要因を利用した物性制御に関する理論的研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 山下 晃一, 東京大学教授 山田 淳夫, 東京大学准教授 牛山 浩, 東京大学准教授 嶺岸 耕, 東京大学准教授 脇原 徹, 東京大学教授 山口 和也University of Tokyo(東京大学

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway

Efficacy of gilteritinib in comparison with alectinib for the treatment of ALK-rearranged non-small cell lung cancer

Gilteritinib is a multitarget tyrosine kinase inhibitor (TKI), approved for the treatment of FLT3-mutant acute myeloid leukemia, with a broad range of activity against several tyrosine kinases including anaplastic lymphoma kinase (ALK). This study investigated the efficacy of gilteritinib against ALK-rearranged non-small cell lung cancers (NSCLC). To this end, we assessed the effects of gilteritinib on cell proliferation, apoptosis, and acquired resistance responses in several ALK-rearranged NSCLC cell lines and mouse xenograft tumor models and compared its efficacy to alectinib, a standard ALK inhibitor. Gilteritinib was significantly more potent than alectinib, as it inhibited cell proliferation at a lower dose, with complete attenuation of growth observed in several ALK-rearranged NSCLC cell lines and no development of drug tolerance. Immunoblotting showed that gilteritinib strongly suppressed phosphorylated ALK and its downstream effectors, as well as mesenchymal-epithelial transition factor (MET) signaling. By comparison, MET signaling was enhanced in alectinib-treated cells. Furthermore, gilteritinib was found to more effectively abolish growth of ALK-rearranged NSCLC xenograft tumors, many of which completely receded. Interleukin-15 (IL-15) mRNA levels were elevated in gilteritinib-treated cells, together with a concomitant increase in the infiltration of tumors by natural killer (NK) cells, as assessed by immunohistochemistry. This suggests that IL-15 production along with NK cell infiltration may constitute components of the gilteritinib-mediated antitumor responses in ALK-rearranged NSCLCs. In conclusion, gilteritinib demonstrated significantly improved antitumor efficacy compared with alectinib against ALK-rearranged NSCLC cells, which can warrant its candidacy for use in anticancer regimens, after further examination in clinical trial settings

光重合型プラスチック暫間充填材の臨床的評価

Fermit, a kind of a visible-light-cured resin, has recently been used as a temporary filling material. This clinical study was done to determine whether or not Fermit was superior to Dura Seal which was previously reported by us in this journal. The prepared cavities were sealed with Fermit for an average of 11.0 days. Fermit was found to have the same properties as Dura Seal, except for many losses of the seal (17.6% of the total) and difficulty in filling

A Clinical Evaluation of a Plastic Temporary Filling Material (Dura Seal®)

Recently Dura Seal, a kind of plastic, has been marketed as a temporary filling material. This clinical study was done to determine whether or not Dura Seal has the properties advertised by the manufacturer. A total of 135 teeth, including 110 vital teeth and 25 pulpless teeth, were prepared for metal inlay cavities. The prepared cavities were sealed with Dura Seal for an average of 12.0 days. Dura Seal was found to be easily removable, to show no pulp damage, and to have considerable mechanical strength

Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations

Background & Aims Biliary tract cancers (BTCs) are clinically and pathologically heterogeneous and respond poorly to treatment. Genomic profiling can offer a clearer understanding of their carcinogenesis, classification and treatment strategy. We performed large-scale genome sequencing analyses on BTCs to investigate their somatic and germline driver events and characterize their genomic landscape. Methods We analyzed 412 BTC samples from Japanese and Italian populations, 107 by whole-exome sequencing (WES), 39 by whole-genome sequencing (WGS), and a further 266 samples by targeted sequencing. The subtypes were 136 intrahepatic cholangiocarcinomas (ICCs), 101 distal cholangiocarcinomas (DCCs), 109 peri-hilar type cholangiocarcinomas (PHCs), and 66 gallbladder or cystic duct cancers (GBCs/CDCs). We identified somatic alterations and searched for driver genes in BTCs, finding pathogenic germline variants of cancer-predisposing genes. We predicted cell-of-origin for BTCs by combining somatic mutation patterns and epigenetic features. Results We identified 32 significantly and commonly mutated genes including TP53 , KRAS , SMAD4 , NF1 , ARID1A , PBRM1 , and ATR , some of which negatively affected patient prognosis. A novel deletion of MUC17 at 7q22.1 affected patient prognosis. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes such as BRCA1 , BRCA2 , RAD51D , MLH1 , or MSH2 were detected in 11% (16/146) of BTC patients. Conclusions BTCs have distinct genetic features including somatic events and germline predisposition. These findings could be useful to establish treatment and diagnostic strategies for BTCs based on genetic information. Lay summary We here analyzed genomic features of 412 BTC samples from Japanese and Italian populations. A total of 32 significantly and commonly mutated genes were identified, some of which negatively affected patient prognosis, including a novel deletion of MUC17 at 7q22.1 . Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes were detected in 11% of patients with BTC. BTCs have distinct genetic features including somatic events and germline predisposition

Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.)

Fruits of persimmon (Diospyros kaki Thunb.) accumulate large amounts of proanthocyanidins (PAs) in the early stages of development. Astringent (A)-type fruits remain rich in soluble PAs even after they reach full-mature stage, whereas non-astringent (NA)-type fruits lose these compounds before full maturation. As a first step to elucidate the mechanism of PA accumulation in this non-model species, we used suppression subtractive hybridization to identify transcripts accumulating differently in young fruits of A- and NA-type. Interestingly, only a few clones involved in PA biosynthesis were identified in A–NA libraries. Represented by multiple clones were those encoding a novel 1-Cys peroxiredoxin and a new member of family 1 glycosyltransferases. Quantitative RT-PCR analyses confirmed correlation of the amount of PAs and accumulation of transcripts encoding these proteins in young persimmon fruits. Furthermore, the new family 1 glycosyltransferase was produced in Escherichia coli and shown to efficiently catalyze galactosylation at 3-hydroxyl groups of several anthocyanidins and flavonols. These findings suggest a complex mechanism of PA accumulation in persimmon fruits