鶴田 宏樹 | ![]() |
ツルタ ヒロキ | |
バリュースクール | |
准教授 | |
農芸化学関係 |
The effect of bacterial colonies expanded into the intervillous spaces on the localization of several lymphocyte lineages was immunohistochemically investigated in two types of mucosa: ordinary mucosa of rat ileum, which consists of mucosa without any mucosal lymphatic tissue; and follicle-associated mucosa (FAM), which accompanies the parafollicular area under the muscularis mucosae in the rat ileal Peyer's patch. The results showed that bacterial colonies in the intervillous spaces induced increased populations of CD8(+) cells in the epithelium of the intestinal villus in ordinary mucosa (IV) and intestinal villus in FAM (IV-FAM). Bacterial colonies in the intervillous spaces were also associated with increased numbers of IgA(+) cells, which were mainly localized in the lamina propria of basal portions of IV and IV-FAM, and with expanded localization of IgA(+) cells into the villous apex in both IV and IV-FAM. Moreover, IgA(+) cells around the intestinal crypts adjacent to IV or IV-FAM were also increased in response to bacterial colonies. In the IV-FAM, but not IV, L-selectin(+) cells, which were found to be immunopositive for TCR alpha beta or CD19, were drastically increased in the lamina propria from the crypt to middle portion of IV-FAM and in the lumen of central lymph vessel of IV-FAM in response to the bacterial colonies in the intervillous spaces. These findings revealed that the expansion of bacterial colonies into the intervillous spaces accompanies the change of histological localization of the lymphocyte lineage in both the ordinary mucosa and FAM.
JAPAN SOC VET SCI, 2019年04月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 81 (4), 555 - 566, 英語, 国内誌[査読有り]
研究論文(学術雑誌)
[査読有り]
研究論文(大学,研究機関等紀要)
計算生命科学は,生命の理解に向けて,近年急速に進展している計算科学と医農工理学分野が融合した学際的研究領域である.様々な研究分野や産業界等への研究の拡がりが期待されており,包括的な基礎知識を習得する機会が求められている.神戸大学計算科学教育センターは,関係諸機関と協力して,遠隔インタラクティブ講義「計算生命科学の基礎」シリーズを2014年から全国に配信を開始し,昨年度は600名の受講登録を受け付けた.本稿では,2017年度に実施した「計算生命科学の基礎IV」と,最近注目されているAIやディープラーニングに焦点を当て特別編として実施したディープラーニングチュートリアルの開催結果について報告する.年々受講者が増え続けており,アンケートでも高評価を得ている..
2018年11月, 大学ICT推進協議会2018年度年次大会論文集, 1 - 4, 日本語研究論文(研究会,シンポジウム資料等)
Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell-derived cellular processes which extended into the LP and the LP cells. The results showed that various cells-including blood capillary, subepithelial stromal cell, and nerve fiber-were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 gm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34(+)CD31(-)alpha SMA(- )stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFR alpha(hi)alpha SMA(+) stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFR alpha(hi)alpha SMA(-) stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34(+)CD31(-)alpha SMA(-)PDGFRa(-/lo) phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. (C) 2018 Wiley Periodicals, Inc.
WILEY, 2018年06月, ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, 301 (6), 1074 - 1085, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Fra a 1 protein in strawberry causes oral allergic syndrome. Over 39 Fra a 1 paralogs have been identified in strawberry genome. Fra a 1.01 is major accumulating protein in edible organs.Strawberry fruits contain allergenic proteins that cause oral allergic syndrome. The hypothesized major allergen is Fra a 1, an ortholog of the birch pollen allergen protein Bet v 1. We organized Fra a 1 genes and analyzed their localizations at the transcriptional and translational levels. In total, 15 new Fra a 1 proteins were identified from the genomic database, increasing the total number of Fra a 1 to 30 proteins encoded by 39 genes. Fra a 1.02 was mostly expressed in receptacles, and Fra a 1.01 in achenes, when analyzed by RNA sequencing. Immunoblotting showed that the Fra a 1.01 protein was broadly accumulated in strawberry organs, while the Fra a 1.02 protein was mostly expressed in receptacles. Recombinant Fra a 1.01 strongly reacted with human IgE. The mRNA and protein expression levels of Fra a 1 did not correlate, indicating the importance of protein levels when evaluating the abundance of allergens in strawberry. Based on the localizations, accumulation levels and reactivity to human IgE, we determined that Fra a 1.01 was the most important allergen, followed by Fra a 1.02, and then other Fra a 1 proteins. The information obtained here will be useful for selecting the target Fra a 1 paralogs when breeding hypoallergenic strawberry.
Springer, 2018年03月, Plant Cell Reports, 37 (3), 411 - 424, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
アクティブラーニングやプロジェクトベーストラーニング(PBL)が高等教育に取り入れられて久しい。特に、近年、卓越大学院大学事業、次世代アントレプレナー育成事業(EDGE-NEXT)など、ワークショップ形式を取り入れた講義を提供する機会が増加している。ここでは、「イノベーション対話ツール」を高等教育におけるワークショップ形式の講義の指南書として活用して実施したワークショップの事例を紹介する。本指南書は、産学連携によるイノベーションを目指した対話を推進するために、平成25年度に文部科学省が慶應義塾大学システムデザイン・マネジメント研究科に委託して作成されたものであるが、これを基にワークショップを設計し、ブレインストーミング、親和図法、バリューグラフを実施したところ、学生らに対して課題解決のプロセスにおける発想力並びに対話力の向上と気づきを提供することがで
神戸大学大学教育推進機構, 2018年03月, 大学教育研究, 26 (26), 27 - 40, 日本語[査読有り]
研究論文(大学,研究機関等紀要)
現代は不確実性の高い社会と呼ばれ、複雑で唯一最適解がない課題が山積されている。さらに科学技術の急激な発展が生活・労働などのヒトが営む活動の環境を加速度的に変化させているのが現代社会の特徴である。このような不確実性の高い社会にこれから出て行く学生は、この社会を変革していく担い手となることが求められている。社会を変革するには、科学技術を基盤とした価値創造、人間を中心にした視点での価値創造による「イノベーション」を引き起こすことが求められる。その担い手が持つ能力は、優れた知識を融合して問題に対峙していける人材である。本論文では、1)現代社会の現状認識とイノベーションを実現する人材の育成の必要性を述べるとともに、2)産業界が求める人材イメージと神戸大学が輩出すべき人材が身につけるべき能力の定義、3)その能力を醸成するための現実社会の問題を題材にした「イノベー
神戸大学大学教育推進機構, 2018年03月, 大学教育研究, 26 (26), 119 - 129, 日本語[査読有り]
研究論文(大学,研究機関等紀要)
Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.
AMER SOC MICROBIOLOGY, 2018年02月, JOURNAL OF VIROLOGY, 92 (3), 英語, 国際誌[査読有り]
研究論文(学術雑誌)
The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4(+) M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4(+) vesicles were frequently detected in the cytoplasms of MV with TLR-4(+) striated borders upstream next to TLR-4(+) M-cells in the FAE of b-LF. These findings suggest that TLR-4(+) MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.
JAPAN SOC VET SCI, 2017年11月, The Journal of Veterinary Medical Science, 79 (11), 1826 - 1835, 英語, 国内誌[査読有り]
研究論文(学術雑誌)
研究論文(研究会,シンポジウム資料等)
The tegument protein U14 of human herpesvirus 6B (HHV-6B) constitutes the viral virion structure and is essential for viral growth. To define the characteristics and functions of U14, we determined the crystal structure of the N-terminal domain of HHV-6B U14 (U14-NTD) at 1.85 angstrom resolution. U14-NTD forms an elongated helix-rich fold with a protruding beta hairpin. U14-NTD exists as a dimer exhibiting broad electrostatic interactions and a network of hydrogen bonds. This is first report of the crystal structure and dimerization of HHV-6B U14. The surface of the U14-NTD dimer reveals multiple clusters of negatively- and positively-charged residues that coincide with potential functional sites of U14. Three successive residues, L424, E425 and V426, which relate to viral growth, reside on the beta hairpin close to the dimer's two-fold axis. The hydrophobic side-chains of L424 and V426 that constitute a part of a hydrophobic patch are solvent-exposed, indicating the possibility that the beta hairpin region is a key functional site of HHV-6 U14. Structure-based sequence comparison suggests that U14-NTD corresponds to the core fold conserved among U14 homologs, human herpesvirus 7 U14, and human cytomegalovirus UL25 and UL35, although dimerization appears to be a specific feature of the U14 group.
PUBLIC LIBRARY SCIENCE, 2016年05月, PLOS PATHOGENS, 12 (5), e1005594, 英語, 国際誌研究論文(学術雑誌)
From previous reports, uptake and accumulation of organic compounds by plant roots are strongly related to water solubility. However, the relation between uptake pathways and water solubility remains unclear. Here, we used confocal laser scanning microscopy to observe the uptake of fluorescent hydrophilic and hydrophobic compounds in the living roots of Cucurbita pepo. We found strong regulation of the uptake of berberine, a hydrophobic compound at the endodermal Casparian strip, as previously reported, and similar regulation was also observed in the pericycle. Berberine was loaded into and transported upward through the protoxylem. Perylene, a highly hydrophobic compound, in contrast, passed through the Casparian strip and accumulated preferentially in the endodermis and pericycle. The results of our solvent extraction suggested that perylene diffused into the non-aqueous phase. Therefore, the uptake pathways for the hydrophilic and hydrophobic compoundss are different. These results offer a new way to understand the uptake of agrochemicals and pollutants and to select host plants for phytoremediation.
日本農薬学会, 2015年, 日本農薬学会誌(Journal of Pesticide Science), 40 (3), 99 - 105, 英語[査読有り]
Consumption of fresh strawberries (Fragaria xananassa) can cause oral allergy syndrome for susceptible individuals. To produce strawberries with low allergen contents a selection of hypoallergenic genotypes and the establishment of culture methods are required. Since several studies have reported that Fra a 1 is a major allergen of strawberry, the expression profiling of the Fra a 1 gene could be an indicator of the allergic potential. In this study, the expression of the Fra a 1 gene was investigated in strawberry during fruit (receptacles and achene) development. The fresh weight and anthocyan content of fruits were also measured to assess their potential correlations with Fra a 1 expression. Fruit of the strawberry were harvested for analyses from a working farm at seven different ripening stages. Real-time PCR revealed the transcript level of the Fra a 1 gene was highest at the early stage of ripening and decreased to approximately 1/70th that level by the red-colored stage. Considering that Fra a 1 belongs to the pathogenesis-related protein 10 family, this result was in line with the report that strawberry fruit may be attacked by oxygen species at the early stage of ripening. Additionally, Fra a 1 gene homologues in Fragaria vesca were surveyed to assess the inducibility of their expression. We found that a gene coding Fra a1 a2 seemed to be expressed in response to stress, and it had major stress-related elements in its promoter region. These results indicate that Fra a 1 expression can be induced by stress, and preharvest treatment to reduce the allergen content will be possible by studying the transcriptional activity of Fra a 1.
INT SOC HORTICULTURAL SCIENCE, 2014年, VII INTERNATIONAL STRAWBERRY SYMPOSIUM, 1049, 323 - 328, 英語[査読有り]
研究論文(国際会議プロシーディングス)
研究論文(その他学術会議資料等)
研究論文(その他学術会議資料等)
研究論文(その他学術会議資料等)
研究論文(国際会議プロシーディングス)
研究論文(国際会議プロシーディングス)
This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.
AMER SOC PLANT BIOLOGISTS, 2013年04月, PLANT PHYSIOLOGY, 161 (4), 2128 - 2135, 英語, 国際誌研究論文(学術雑誌)
研究論文(大学,研究機関等紀要)
Complexing of green tea catechins with food constituents and their hydrolysis by tannase-producing Lactobacillus plantarum strains, were investigated. Our observations indicated that 1) epigallocatechin gallate (EGCg) and other catechin galloyl esters bound with food ingredients (i.e., proteins) to form a complex that is likely to be unabsorbable through the intestinal wall, whereas most catechins not esterified with gallic acid (GA) remain in free form, not complexing with food ingredients; 2) tannase activity of L. plantarum is strain dependent, possibly grouped into those with high tannase activity hydrolyzing EGCg to epigallocatechin and GA and those with the low activity; and 3) L. plantarum strains with high tannase activity are capable of hydrolyzing not only intact EGCg but also EGCg and other catechin galloyl esters complexed with dietary proteins to free non-galloyl ester catechins and GA. The evidence suggests that L. plantarum with high tannase activity, if it colonizes the human intestine, would release free non-galloyl-ester catechins and GA that are readily absorbed through the human intestinal epithelia from the complexes, thereby ensuring maximum delivery of the bioactive polyphenols of green tea to the host.
2012年, Bioscience of microbiota, food and health, 31 (2), 27 - 36, 英語, 国内誌[査読有り]
研究論文(学術雑誌)
Crystals of beta-1,3-xylanase (1,3-beta-D-xylan xylanohydrolase; EC 3.2.1.32) from Thermotoga neapolitana strain DSM 4359 with maximum dimensions of 0.2 x 0.1 x 0.02 mm were grown using the sitting-drop vapour-diffusion method at 293 K over 24 h. The crystals diffracted to a resolution of 1.82 angstrom, allowing structure determination. The crystals belonged to space group P2(1), with unit-cell parameters a = 39.061, b = 75.828, c = 52.140 angstrom; each asymmetric unit cell contained a single molecule.
INT UNION CRYSTALLOGRAPHY, 2011年07月, Acta Crystallographica Section F-Structural Biology and Crystallization Communications, 67 (Pt 7), 779 - 781, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
The polymorphism of ORFs encoding putative cell-surface adhesins was investigated in Bifidobacterium longum subsp. longum. Firstly, we performed a PCR assay targeting 15 ORFs encoding putative adhesion proteins, which included 8 ORFs with a sortase targeting LPXTG motif, in 42 strains of different pulsotypes isolated from fecal samples from 12 human individuals. We found a variability in the presence of an ORF, BL0675, which encodes a putative fimbrial subunit protein. We sequenced ORFs corresponding to BL0675 in the 42 strains and adjacent ORFs corresponding to BL0674 and BL0676. The results indicated that ORFs corresponding to BL0675 were highly polymorphic with five variant types (i.e. A-, B-, C-, D-, and E-types). Meanwhile, BL0674 and BL0676, which encode an additional putative fimbrial subunit protein and a fimbrial-associated sortase-like protein, were highly conserved. Subsequent quantitative polymerase chain reaction (qPCR) assays targeting the variant types in 89 human fecal samples revealed that A-type was the most commonly distributed (74.2%), followed by B-type (59.6%), D-type (31.5%), E-type (32.6%) and C-type (5.6% prevalence). Since BL0675 is considered to be a fimbrial protein with glycoprotein-binding ability, the proteins encoded by the five variant types of BL0675 may have specific binding properties to various carbohydrate structures expressed on the human intestinal wall, thereby allowing B. longum to colonize the intestine in a host-specific manner.
SPRINGER, 2011年03月, ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, 99 (3), 457 - 471, 英語, 国際誌研究論文(学術雑誌)
Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 mu g/ml in tertiary peptide and from 10 to 40 mu g/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 mu g/ml in the peptide with tertiary structure and about 10 mu g/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.
SPRINGER, 2011年01月, EXPERIMENTAL AND APPLIED ACAROLOGY, 53 (1), 71 - 77, 英語, 国際誌研究論文(学術雑誌)
Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH2-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K-i value of 6.2 +/- 0.55 nM. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn(32)-Met(38) and Gly(97)-Leu(103), in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val(36)-centerd hydrophobic cluster within the Asn(32)-Met(38) region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 degrees C was only partial and reversible. A tripeptide, Ile(35)-Val(36)-Tyr(37), in the Asn(32)-Met(38) region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年09月, JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (39), 29797 - 29807, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
The crystal structure of a cold-active alkaline phosphatase from a psychrophile, Shewanella sp. (SCAP), was solved at 2.2 angstrom. A refined model showed a homodimer with six metal-ligand sites. The arrangement of the catalytic residues resembled those of alkaline phosphatases (APs), suggesting that the reaction mechanism of SCAP was fundamentally identical to those of other Alps. SCAP had two distinct structural features: (i) a loop with Arg122 that bound to the phosphate moiety of the substrate suffered no constraints from the linkage to other secondary structures, and (ii) Mg3-ligand His109 was considered to undergo repulsive effect with neighboring Trp228. The local flexibility led by these features might be an important factor in the high catalytic efficiency of SCAP at low temperatures.
TAYLOR & FRANCIS LTD, 2010年01月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74 (1), 69 - 74, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917 T. This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5 alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases. (C) 2008 Elsevier GmbH. All rights reserved.
ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2008年09月, SYSTEMATIC AND APPLIED MICROBIOLOGY, 31 (4), 269 - 277, 英語, 国際誌研究論文(学術雑誌)
The cold-active protein tyrosine phosphatase found in psychrophilic Shewanella species exhibits high catalytic efficiency at low temperatures as well as low thermostability, both of which are characteristics shared by many cold-active enzymes. The structure of cold-active protein tyrosine phosphatase is notable for the presence of three hydrophobic sites (termed the CA, Zn-1 and Zn-2 sites) behind the loop structures comprising the catalytic region. To identify the structural components responsible for specific enzyme characteristics, we determined the structure of wild-type cold-active protein tyrosine phosphatase at high resolution (1.1 angstrom) and measured the catalytic efficiencies of enzymes containing mutations in the three hydrophobic sites. The bulkiness of the amino acid side chains in the core region of the Zn-1 site strongly affects the thermostability and the catalytic efficiency at low temperatures. The mutant enzyme I115M possessed a higher k(cat) at low temperatures. Elucidation of the crystal structure of I115M at a resolution of 1.5 angstrom revealed that the loop structures involved in retaining the nucleophilic group and the acid catalyst are more flexible than in the wild-type enzyme.
WILEY, 2008年09月, FEBS JOURNAL, 275 (17), 4317 - 4328, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Phospholipase D, with a molecular mass of 64 kDa, was purified from the psychrophile, Shewanella sp. The enzyme showed maximal activity at pH 7.8 and 40 degrees C in the presence of the Ca2+-ion, and its activity at 10 degrees C was 6.5% of maximum. The enzyme exhibited high activity to the non-micelle form of phosphatidylcholine in an aqueous solution containing water miscible alcohols such as methanol, ethanol, iso-propanol, and n-propanol. Nucleotide sequencing of the enzyme gene yielded a deduced amino acid sequence, which showed 36.2% identity to that of Streptomyces chromofuscus phopsholipase D alone. The low sequence similarity to other phopsholipase D enzymes suggests that the purified enzyme might be a novel phospholipase D.
TAYLOR & FRANCIS LTD, 2007年10月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71 (10), 2534 - 2542, 英語, 国際誌研究論文(学術雑誌)
[査読有り]
研究論文(学術雑誌)
The cold-active protein-tyrosine phosphatase (CAPTPase) of a psychrophile, Shewanella sp., shows high catalytic activity below 20 degrees C. The catalytic residue of CAPTPase is histidine, as opposed to the cysteine of known protein-tyrosine phosphatases (PTPases), and the enzyme protein has three amino acid sequences, Asp-Xaa-His, Gly-Asp-Xaa-Xaa-Asp-Arg and Gly-Asn-His-Glu, that are observed in many protein-serine/threonine phosphatases (PS/TPases). We have determined the crystal structures of CAPTPase at 1.82 angstroms and the enzyme bound with a phosphate ion at 1.90 angstroms resolution using X-ray crystallography and the multiple isomorphous replacement method. The final refined models are comprised of 331 amino acid residues, two metal ions, 447 water molecules, and an acetate or phosphate ion in an asymmetric unit. The enzyme protein consists of three beta-sheets, termed Sheet I, Sheet I', and Sheet II, and 14 alpha-helices. The CAPTPase has a different overall structure from known protein-tyrosine phosphatases. The arrangement of two metal ions, a phosphate ion and the adjacent amino acid residues in the catalytic site of CAPTPase is identical to that of PS/TPases. Thus, it was confirmed that the CAPTPase was a novel PTPase with a conformation similar to the catalytic site of PS/TPase. We speculate that the hydrophobic moiety around the catalytic residue of CAPTPase might play an important role in eliciting high activity at low temperature.
JAPANESE BIOCHEMICAL SOC, 2005年01月, Journal of biochemistry, 137 (1), 69 - 77, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Protein-tyrosine phosphatase [EC 3.1.3.48] from a psychrophile, Shewanella sp. shows high activity at low temperatures and has the conserved amino acid sequence of protein-Ser/Thr-phosphatases. Site-directed mutagenesis with the conserved amino acid residues indicated that His148 could be important as a general acid catalyst and Asp115 assists the protonation with His148 of the leaving group of a substrate, and that Asp76 and Asp112 were involved in binding to magnesium ions.
TAYLOR & FRANCIS LTD, 2004年02月, Bioscience, biotechnology, and biochemistry, 68 (2), 440 - 3, 英語, 国際誌研究論文(学術雑誌)
A procedure was established for expression and purification of abundant recombinant cold-active protein-tyrosine-phosphatase (RCPTPase), which showed identical enzymatic characteristics to the native enzyme (NCPTPase). The purified RCPTPase showed high catalytic activity at low temperature and maximal activity at 30 degrees C. RCPTPase has a thermodynamic characteristic in that its activation enthalpy was determined to be low, 4.3 kcal/mol, at temperatures below 19.3 degrees C, where the Arrhenius relationship exhibited an inflection point, in comparison with 20.3 kcal/mol above 19.3 degrees C. Also, the thermostability, DeltaG(water), of the catalytic site in the RCPTPase molecule was increased with a decrease in temperature. It was considered that cold-active protein-tyrosine-phosphatase could maintain its catalytic site in a stable conformation for eliciting high catalytic activity with low activation enthalpy at low temperature.
2003年02月, Journal of biochemistry, 133 (2), 225 - 30, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
Cold-active protein-tyrosine phosphatase (PTPase) of Shewanella sp. was expressed, purified and crystallized using the hanging-drop vapour-diffusion method at two different pH values (4.6 and 8.5). Both crystals are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.4, b = 76.8, c = 81.0 A (pH 8.5) and a = 57.1, b = 77.0 and c = 81.5 A (pH 4.6). Diffraction data to 1.82 A for the pH 8.5 crystal and 2.33 A for the pH 4.6 crystal were collected on a multiwire area detector using a rotating-anode X-ray generator.
BLACKWELL MUNKSGAARD, 2002年09月, Acta crystallographica. Section D, Biological crystallography, 58 (Pt 9), 1465 - 6, 英語, 国際誌[査読有り]
研究論文(学術雑誌)
A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Blosci. Blotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.
TAYLOR & FRANCIS LTD, 2002年04月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66 (4), 754 - 761, 英語[査読有り]
研究論文(学術雑誌)
Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3.1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.
JAPANESE BIOCHEMICAL SOC, 2000年01月, JOURNAL OF BIOCHEMISTRY, 127 (1), 143 - 149, 英語[査読有り]
研究論文(学術雑誌)
Phosphatase I purified from a psychrophile (Shewanella sp,) [Tsuruta et al. (1998) J. Biochem, 123, 219-225] dephosphorylated O-phospho-L-tyrosine and phospho-tyrosyl residues in phosphorylated poly(Glu(4),Tyr(1)) random polymer (polyEY) and phosphorylated myelin basic protein (MBP) but not phosphoseryl and/or phosphothreonyl residues in phosphorylated histone H1, casein and phosphorylase a, indicating that the enzyme showed protein-tyrosine-phosphatase (PTPase, EC 3.1.3.48)-like activity in vitro. The enzyme was remarkably inhibited by diethylpyrocarbonate (DEPC), monoiodoacetic acid (MIAA), and monoiodoacetamide (MIAM). Binding of 1 mol of DEPC to 1 mol of the enzyme caused complete inhibition of the enzyme; and 0.88 mol of l-carboxymethylated histidine per mole of the enzyme was found when 90% of enzyme activity was lost by modification with C-14-MIAA. These results indicated that this psychrophilic enzyme was a PTPase-like enzyme with histidine as its catalytic residue.
JAPANESE BIOCHEMICAL SOC, 1999年04月, JOURNAL OF BIOCHEMISTRY, 125 (4), 690 - 695, 英語[査読有り]
研究論文(学術雑誌)
The phosphatase of a psychrophile (Shewanella sp.) was purified by ammonium sulfate fractionation, followed by sequential column chromatographies. The purified enzyme was electrophoretically homogeneous on native- and SDS-PAGE. Its molecular weight was 41,826 by its amino acid composition. The enzyme had its optimal pH for the activity at 9.8, and a broad substrate specificity to dephosphorylate ATP, pyrophosphate, glycerophosphate, and so on. Its activity was increased by metal ions including Mg2+, Mn2+, and Co2+. The maximal activity was observed at 40 degrees C, and the enzyme at 0 degrees C showed 39% of activity at 40 degrees C. The enzyme, however, tended to lose its activity at 20 degrees C and pH 9.8. These results indicated that purified enzyme was an alkaline phosphatase with characteristics; high catalytic efficiency at low temperature and gradual inactivation at an intermediate temperature.
TAYLOR & FRANCIS LTD, 1998年11月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 62 (11), 2246 - 2250, 英語[査読有り]
研究論文(学術雑誌)
The phosphatase of a psychrophile was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, butyl-Cellulofine, Sephacryl S-100, and Mono-Q columns, The purified enzyme preparation was found to be electrophoretically homogeneous on native-and SDS-PAGE, and its molecular mass was determined to be 38.4 kDa by MALDI-TOF mass spectrometry, Maximal activity was observed at 30 degrees C and pH 6.0, Furthermore, the activity of this enzyme at 0 and 5 degrees was 27 and 28%, respectively, of that at 30 degrees C, The enzyme was stable in the pH range of 6.0 to 8.0 and up to 20 degrees C. The enzyme was affected by metal ions; the activity was enhanced by Mg2+ and Ca2+ ions, but depressed by Zn2+ ions, Analysis of the amino acid composition indicated that this phosphatase contains no S-S bond, and only a few prolyl residues necessary to retain the rigid structure of a protein molecule, The phosphatase shows typical features of a cold enzyme; high catalytic activity at low temperature and rapid inactivation at an intermediate temperature.
JAPANESE BIOCHEMICAL SOC, 1998年02月, JOURNAL OF BIOCHEMISTRY, 123 (2), 219 - 225, 英語[査読有り]
研究論文(学術雑誌)
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