Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC)

Sodium-metal-promoted reductive 1,2-syn-diboration of alkynes with reduction-resistant trimethoxyborane

Reductive 1, 2-diboration of alkynes has been accomplished by means of sodium dispersion in the presence of trimethoxyborane as a reduction-resistant boron electrophile. Two boron moieties can be introduced onto alkynes with excellent syn selectivity to afford the corresponding (Z)-1, 2-diborylalkenes. Bis(borate) species generated in situ can be involved in one-pot Suzuki-Miyaura arylation, formal arylboration of alkynes thus being executed

江戸時代の唐画

学位の種別:課程博士University of Tokyo(東京大学

Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon

C-Glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-Glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C-glycosylflavonoidshowever, the genes encoding for enzymes that catalyze C-glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C-glucosyltransferase gene from the dicot plant Fagopyrum esculentumM. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C-glucosylation of 2-hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C-glucosylation activity towards 2-hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2,4,6-trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C-glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.ArticlePLANT JOURNAL. 80(3):437-448 (2014)journal articl

真景図を写す : 武元登々庵をめぐる画家 大西圭斎と大原東野

漢詩人、書家として知られる武元登々庵（1767 ～ 1818）は寛政11 年（1799）から翌年にかけて画家大西圭斎とともに奥州を旅した。登々庵は圭斎が描いた「真景図」をもとに「登々庵奥遊詩画巻」（広島県立歴史博物館、重要文化財「菅茶山関係資料」）を別の画家に描かせ、儒学者菅茶山に見せた。茶山は「登々庵奥遊詩画巻」とは別に圭斎の「真景図」を写させて画帖の「登々庵奥遊詩画」（広島県立歴史博物館、重要文化財「菅茶山関係資料」）を作っている。茶山の弟子で尾道の豪商でもある松本孟執（泉屋治右衛門）が「登々庵奥遊詩画巻」を自ら写したのが「奥秘三勝」（岡山県立博物館）である。登々庵の周辺では実景に接しない画家によって登々庵の実体験に結びつく作品が作られていた。「真景」と題された作品を我々は画家の実体験と結びつけがちだが、画家ではなく注文主、賛者にとっての「真景」を想定できる。実景に接しない画家による実景図が多数作られたことを思えば、「真景」でも同様に画家の実体験を伴わない「真景図」が考えられる。From 1799 to 1800, the famous poet and calligrapher Takemoto Totoan (1767-1818) travelled around Oshu in the Tohoku region with the painter Onishi Keisai. Totoan later commissioned another painter to create the hand scroll Totoan Oyu Shigakan (Poems and Paintings from Totoan’s Journey to Oshu ; Hiroshima Prefectural Museum of History, Koyo Sekiyo bunko, Kan Chazan Collection), which depicts four views from the journey based on true-view sketches by Keisai. Totoan showed this scroll to Kan Chazan, a famous Confucian scholar, who then had another painter make an album after Keisai’s sketches that bears the same title as the scroll: Totoan Oyu Shiga (Poems and Paintings from Totoan’s Journey to Oshu; Hiroshima Prefectural Museum of History, Koyo Sekiyo bunko,Kan Chazan Colleciton). Moreover, Chazan’s pupil, Matsumoto Moshu, who was also known as the wealthy merchant Izumiya Jiemon of Onomichi, painted OhiSansho (Three Secret Views of Oshu ; Okayama Prifectural Museum) based on the scroll. Thus, a number of painters were depicting views that Totoan, not they, had witnessed

Structural difference due to intramolecular stacking interactions in dinuclear rhodium(III) complexes [{Rh(η5-C5Me5)(L)}2]n+containing pyrimidine-2-thionate and related ligands

Self-assembling reactions between [Rh(η5-C5Me5)(H2O)3]2+and pyrimidine-2-thionate(pymt) or related ligands[L; mpymt = 4-methyl-pyrimidine-2-thionate(1-), dmpymt = 4,6-dimethylpyrimidine-2-thionate(1-), apymt = 4-aminopyrimidine-2-thionate(1-), dapymt = 4,6-diaminopyrimidine-2-thionate(1-), or mpol = 2-sulfanyl-3-pyridinolate(2-)] were carried out and the products characterized by UV/vis, NMR spectroscopy, electrospray ionization mass spectrometry, and crystal structure analysis. All products are dinuclear rhodium(III) complexes of [{Rh(η5-C5Me5)(L)}2]n+: three crystal structures with mpymt, dmpymt and mpol were determined. The mpymt and dmpymt ligands co-ordinate through a 1κ2N,S:2κS mode and the two pyrimidine rings are located in cis position,whereas mpol adopts a five-membered chelating mode with 1κ2S,O:2κS and the two pyrimidine rings are located in trans position. Such structural difference can reasonably be explained by the　intramolecular stacking interaction between the two bridging ligands

Body mass index and colorectal cancer risk : A Mendelian randomization study

Traditional observational studies have reported a positive association between higher body mass index (BMI) and the risk of colorectal cancer (CRC). However, evidence from other approaches to pursue the causal relationship between BMI and CRC is sparse. A two-sample Mendelian randomization (MR) study was undertaken using 68 single nucleotide polymorphisms (SNPs) from the Japanese genome-wide association study (GWAS) and 654 SNPs from the GWAS catalogue for BMI as sets of instrumental variables. For the analysis of SNP-BMI associations, we undertook a meta-analysis with 36 303 participants in the Japanese Consortium of Genetic Epidemiology studies (J-CGE), comprising normal populations. For the analysis of SNP-CRC associations, we utilized 7636 CRC cases and 37 141 controls from five studies in Japan, and undertook a meta-analysis. Mendelian randomization analysis of inverse-variance weighted method indicated that a one-unit (kg/m2) increase in genetically predicted BMI was associated with an odds ratio of 1.13 (95% confidence interval, 1.06-1.20; P value <.001) for CRC using the set of 68 SNPs, and an odds ratio of 1.07 (1.03-1.11, 0.001) for CRC using the set of 654 SNPs. Sensitivity analyses robustly showed increased odds ratios for CRC for every one-unit increase in genetically predicted BMI. Our MR analyses strongly support the evidence that higher BMI influences the risk of CRC. Although Asians are generally leaner than Europeans and North Americans, avoiding higher BMI seems to be important for the prevention of CRC in Asian populations