コウネツセイ サイキン Thermus thermophilus ムキ ピロリンサン カスイ ブンカイ コウソ ノ error-prone PCR ヘンイ ドウニュウ ニヨル ネツ ギョウシュウ ヨクセイ

Thermus thermophilus Inorganic pyrophosphatase (E.C. 3.6.1.1., Tth PPase) exhibits high thermostability, but thermal aggregation was observed on heating above 85 °C. In addition, we reported that sole cysteine in C-terminal region plays a key role in the thermostability and thermal aggregation of Tth PPase [Kohaku, Y. et al. (2008) Natl.Sci.Res., 22, 75-84]. In this study, we approached the suppression of its thermal aggregation by error-prone PCR mutagenesis of whole molecule or C-terminal region. Firstly, we obtained thermostable four variants (Q119H/L162F, L162F, K173E and K159E/A170T) by error-prone PCR mutagenesis. Moreover, we examined thermostabilities of four variants in terms of the enzyme activity, tertiary and quaternary structure. Although conformation and quaternary structure of four variants were almost the same as those of wild type enzyme in native state, K173E and K159E/A170T variants showed higher thermostabilities than wild type in tertiary and quaternary structure. In particular, thermal aggregation of these two variants would be suppressed after heating at 85°C. Therefore, it was suggested that Lys159 and Lys173 in the molecular surface of C-terminal region may contribute to the formation of thermal aggregation of Tth PPase

Dealkylation of alkyl polycyclic aromatic hydrocarbon over silica monolayer solid acid catalyst

Dealkylation of alkylnaphthalene, as a model of alkyl polycyclic aromatic hydrocarbon compounds in heavy oils, proceeded selectively on a silica monolayer solid acid catalyst. The activity was generated by the deposition of silica on alumina with generation of Brønsted acidity. The activity and Brønsted acid amount showed the maximum where the monolayer covered the surface, indicating that the Brønsted acid site generated on the silica monolayer was the active species. The activity and selectivity on the silica monolayer were high compared to other aluminosilicate catalysts, and high activity was observed even after calcination at 973–1173K

カンケイ ドウブツ アオゴカイ Perinereis aibuhitensis キョダイ ヘモグロビン ノ グロビンサ ニ フクマレル ケウナ SSケツゴウ

The extracellular hemoglobin (Hb) from the polychaete Perinereis aibuhitensis consists of four types of 144 globins and two types of 36 linker chains，having a molecular mass of about 3,500 kDa. There are two types of globin subunits: monomer chain a and disulfide-bondedt rimer AbB. The amino acid sequences of the four globin chains (a，A ，b ，B ) were already reported，previously (Yamanaka，M. et al. Natural Science Research of Tokushima University, 19, 63-92, 2005). The site of disulfide bonds in the globin subunits have been investigated. Each globin chain contains an intrachain disulfide bond between N-terminal and C-terminal Cys residues. In addition，the interchain disulfide bonds were found between chains A and b，and b and B. Therefore，it is elucidated that the chain b is situated at the center of disulfide-bonded trimer，such as A-b-B.The sites of disulfide-bonds determined all could be suitably fitted to the tree dimensional structure of each subunit in a model without stretching or twisting. It was also confirmed that there is no free Cys residue in Peinereis globins. The positions of Cys residues of Perinereis globin sequences were compared wIth those of other 27 chains derived from the homologous Hbs. Among 31 sequences，Cys residues were distributed in six sites. The sites 1 and 2 are located at the N-terminal region of amino acid sequences，the sItes 3 and 4 at the central region，and the sites 5 and 6 at C-terminal region. Furthermore，the Cys distribution was categorized into eight patterns. Perinereis Hb has four patterns I，IV，VII，VIII，be ing lack of the central sites 3 and 4. It should be noted that the pattern II includes the unique globins from Hbs of Lamellibrachia，Riftia and Oligobrachia that carry H2S to the symbiotic bacteria，suggesting that these globin chains might carry H2S in vivo. The phylogenetic tree of 31 globin chains derived from the giant Hbs is divided into two families A and B， as already poited out by us Previously. The family A indudes pattern I-V，whereas the family B includes V- VIII

Effect of Cys168 substitutions on the Thermostability and the Thermal Aggregation of Thermus thermophilus Inorganic Pyrophosphatase.

Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) is comprised of homohexamer，and exhibits high thermostability. However，the thermal aggregation containing the cross-linked dimer was observed after heating above 85℃. Therefore，we focused on the sole cysteine (Cys168) in C-terminalregion，and evaluated the effects of substitutions at this position on thermostability and thermal aggregation of Tth PPase. Firstly，we prepared the four Cys168-substituted variants (C168A，L ，1，and F) by site-directed mutagenesis. Although all variants formed hexamer in native state，C168A variant exhibited the highest thermostabilities for the enzyme activity and quatemary structure in wild type and all variants，while the other variants decreased them drastically as the side chain at the 168 position was much more bulky and hydrophobic in Tth PPase. Moreover， suppression of thermal aggregation for C168A variant was observed in the ANS fluorescence experiments. Therefore，we suggest that the small volume and less hydrophobicity of side chain at 168 position may contribute to the conformational thermostability, and substitution with Ala is the most suitable for thermostabilization and suppression ofthermal aggregation of Tth PPase

Reassociation of annelid giant hemoglobin from the polychaete Perinereis aibuhitensis

Annelid extracellular hemoglobin (Hb) is a supramolecule with molecular mass of ～3,500kDa. The giant Hb consists of 12 subassemblies (globin dodecamers, D) and 18 homodimeric linkers (L) of non-globin chain. The globin dodecamer and linker were isolated from the polychaete Perinereis aibuhittensis Hb separately. Subsequently, these two components were mixed in the presence of 1M urea at a neutral pH to reform a whole molecule of Hb. At first L was refined by reverse phase chromatography in organic solvent. On the other hand, Perinereis Hb was incubated in 4M urea solution at 4°C for 5 min, and applied to two amphoteric ion-exchange resin column to remove L stick to the resin, and to isolate only D. The eluate was condensed and subjected to gel filtration. As a result, an ingredient of molecule mass ～210 kDa, that is D, was provided in high yield. When D and L were mixed in the molar ratio of approximately 1:1 in 50mM phosphate buffer (pH 7.2) in the presence of 1 M urea at room temperature, most of the proteins met to natural Hb size again within about 20 hours. Furthermore, similar experiments were performed in 50 mM Tris-HCl buffer (pH 7.2) containing 1 M urea in the presence of 1 mM CaCl2 or 1mM EDTA. It was observed that the reassociation was affected substantially by the presence of Ca2+. In conclusion, the homodimeric linkers have the key role to form the gigantic Hb

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

FLOOD DAMAGES OF SOCIAL WELFARE FACILITIES ALONG THE OPPE RIVER DUE TO TYPHOON HAGIBIS IN 2019 AND EFFECT OF FACILITY CHARACTERISTICS ON BUSINESS CONTINUITY

令和元年東日本台風では越辺川右岸0.0k付近の堤防決壊により，近くの高齢者施設（1法人2施設）や障害者施設（1法人6施設）が甚大な被害を受けた．被災した2法人を対象に，被害状況，避難行動，避難確保計画，被災後の業務再開状況等についてインタビュー調査を行った．また，浸水痕跡調査を実施した上で，破堤に伴う川越市内の氾濫進展状況を数値シミュレーションにより再現し，証言内容の検証を行った．高齢者施設と障害者施設の利用者の身体的特性や，避難方法，避難生活などの情報を施設特性に注目して整理し，施設特性を踏まえた避難確保計画と長期避難を想定した業務継続計画を検討しておくことの重要性について明らかにした．In Typhoon Hagibis in 2019, a elderly welfare facility and a welfare facility of the mentally handicapped persons suffered severe flood damage by the collapse of an embankment at the 0km point on the Oppe River. Interview surveys were conducted on these social welfare facilities regarding the damage situation, emergency response, and resumption of operations after the disaster. The inundation trace surveys and flood inundation analysis were conducted to deeply understand the actual damage situation. From these results, we focus on the difference between the characteristics of the welfare facilities for the elderly and the disability persons, extract the issues, and consider the points to consider when formulating an evacuation securement plan and a business continuity plan for social welfare facilities