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41.
Setsu Nakae Shigeaki Ito Mariko Higa Takeshi Senoura Jun Wasaki Atsushi Hijikata Masafumi Shionyu Susumu Ito Tsuyoshi Shirai 《Journal of molecular biology》2013
The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-d-mannosyl-d-glucose and phosphate, and the product d-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases. 相似文献
42.
Mari Kawaji Shingo Kaneko Ryunosuke Tateno Yuji Isagi Tsuyoshi Yoneda 《Conservation Genetics》2009,10(4):1049-1051
Quercus miyagii is an endemic tree species in the Ryukyu Islands, Japan. We isolated and characterized 15 microsatellite loci in this species.
The number of alleles ranged from 2 to 16 and expected heterozygosities from 0.07 to 0.92. This set of markers is potentially
useful to investigate the genetic structure, gene flow, and the biogeographic history of Q. miyagii in the Ryukyu Islands, Japan. 相似文献
43.
Qingzhu Yuan Tsuyoshi Adachi Shinji Takenak Shuichiro Murakami Machiko Tanak Kenji Aoki 《生物工程学报》2008,24(7):1221-1227
Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose. 相似文献
44.
Src kinase activity is essential for osteoclast function 总被引:21,自引:0,他引:21
Miyazaki T Sanjay A Neff L Tanaka S Horne WC Baron R 《The Journal of biological chemistry》2004,279(17):17660-17666
Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts. 相似文献
45.
Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes 总被引:1,自引:0,他引:1
Goto T Lee JY Teraminami A Kim YI Hirai S Uemura T Inoue H Takahashi N Kawada T 《Journal of lipid research》2011,52(5):873-884
Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs. 相似文献
46.
Ramida Yuwadee Watanapokasin Anantabhathra Boonyakamol Supawadee Sukseree Aungkana Krajarng Thanet Sophonnithiprasert Sungwan Kanso Tsuyoshi Imai 《Biodegradation》2009,20(3):411-418
Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates,
glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l
was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual
glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess
decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which
was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization
occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be
the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water. 相似文献
47.
Mariejoy Therese Jumawid Tsuyoshi Takahashi Toshimasa Yamazaki Hiroshi Ashigai Hisakazu Mihara 《Protein science : a publication of the Protein Society》2009,18(2):384-398
The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties. 相似文献
48.
Naoshi Yamamoto Sayaka Ohrui Takahiro Okada Tsuyoshi Saitoh Noriki Kutsumura Yasuyuki Nagumo Yoko Irukayama-Tomobe Yasuhiro Ogawa Yukiko Ishikawa Yurie Watanabe Daichi Hayakawa Hiroaki Gouda Masashi Yanagisawa Hiroshi Nagase 《Bioorganic & medicinal chemistry》2019,27(8):1747-1758
Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX1R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX1R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX1R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives. 相似文献
49.
Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk 总被引:2,自引:0,他引:2
Mogi T Hatakeyama K Taguchi T Wake H Tanaami T Hosokawa M Tanaka T Matsunaga T 《Biosensors & bioelectronics》2011,26(5):1942-1946
This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis. 相似文献
50.
Tohru Kobayashi Yasushi Kageyama Nobuyuki Sumitomo Katsuhisa Saeki Tsuyoshi Shirai Susumu Ito 《World journal of microbiology & biotechnology》2005,21(6-7):961-967
Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not
found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered
into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused
the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal
inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This
result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the
highly alkaline M-protease. 相似文献