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21.
Shikimate kinase from Phaseolus mungo seedlings was partiallypurified by DEAEcellulose, hydroxyapatite and Sephacryl S-200column chromatographies. The activity was completely inhibitedby EDTA and the requirement for Mg2+ could be partially replacedby Mn2+, Ca2+; Co2+ and Cd2+. Sulfhydryl inhibitor did not inhibitthe enzyme activity. The apparent Km values for shikimic acidand ATP at pH 8.6 were 0.25 mM and 0.38 mM, respectively. Theactivity appeared to be maximal at pH 8.69.0. Shikimate-3-phosphateand ADP inhibited the activity slightly. Aromatic amino acids,quinic acid and dehydroquinic acid had no significant effecton the activity. (Received January 11, 1979; ) 相似文献
22.
Fibrillin‐1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs‐like) 6β is one of the fibrillin‐1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6β in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6β and examined their interactions with fibrillin‐1 assembly. Pull‐down assay of the ADAMTSL6β deletion mutants and fibrillin‐1 protein revealed that ADAMTSL6β binds to fibrillin‐1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin‐1 matrix assembly was enhanced in MG63 cells, expressing full‐length ADAMTSL6β, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3‐ADAMTSL6β form showed enhanced assembly formation, Δ TSP2‐ADAMTSL6β form did not enhance that, indicating the difference between Δ TSP2‐Δ TSP3 has a critical role for fibrillin‐1 assembly. As the difference of Δ TSP2‐Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6β influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6β in the process of microfibril formation. 相似文献
23.
Masahiro Nakamori Tetsuya Takahashi Tomokazu Nishikawa Yu Yamazaki Takashi Kurashige Hirofumi Maruyama Koji Arihiro Masayasu Matsumoto 《PloS one》2013,8(11)
Background
Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.Methods
Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.Results
GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.Conclusions
These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins. 相似文献24.
Daiki Miki Hidenori Ochi Atsushi Takahashi C. Nelson Hayes Yuji Urabe Hiromi Abe Tomokazu Kawaoka Masataka Tsuge Nobuhiko Hiraga Michio Imamura Yoshiiku Kawakami Hiroshi Aikata Shoichi Takahashi Norio Akuta Fumitaka Suzuki Kenji Ikeda Hiromitsu Kumada Yoshiyasu Karino Joji Toyota Tatsuhiko Tsunoda Michiaki Kubo Naoyuki Kamatani Yusuke Nakamura Kazuaki Chayama 《PloS one》2013,8(12)
Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P
combined = 3.59 × 10−16, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype. 相似文献
25.
Hiroko Iijima Atsuko Watanabe Haruna Sukigara Tomokazu Shirai Akihiko Kondo Takashi Osanai 《Biotechnology and bioengineering》2020,117(6):1649-1660
Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L−1·day−1, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria. 相似文献
26.
Naoko Hattori Tohru Niwa Kana Kimura Kristian Helin Toshikazu Ushijima 《Nucleic acids research》2013,41(15):7231-7239
Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications. 相似文献
27.
Takahito Tamai Osamu Yamaguchi Shungo Hikoso Toshihiro Takeda Manabu Taneike Takafumi Oka Jota Oyabu Tomokazu Murakawa Hiroyuki Nakayama Yoshihiro Uno Kyoji Horie Kazuhiko Nishida Nahum Sonenberg Ajay M. Shah Junji Takeda Issei Komuro Kinya Otsu 《The Journal of biological chemistry》2013,288(14):10176-10187
Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb−/−). Rheb−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb−/− was lower than that in the control (Rheb+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb+/+ mice but not in Rheb−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb−/− hearts during the neonatal period. Rheb−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period. 相似文献
28.
Tomokazu Ito Jumpei Iimori Sayuri Takayama Akihito Moriyama Ayako Yamauchi Hisashi Hemmi Tohru Yoshimura 《Journal of bacteriology》2013,195(24):5439-5449
Escherichia coli YggS is a member of the highly conserved uncharacterized protein family that binds pyridoxal 5′-phosphate (PLP). To assist with the functional assignment of the YggS family, in vivo and in vitro analyses were performed using a yggS-deficient E. coli strain (ΔyggS) and a purified form of YggS, respectively. In the stationary phase, the ΔyggS strain exhibited a completely different intracellular pool of amino acids and produced a significant amount of l-Val in the culture medium. The log-phase ΔyggS strain accumulated 2-ketobutyrate, its aminated compound 2-aminobutyrate, and, to a lesser extent, l-Val. It also exhibited a 1.3- to 2.6-fold increase in the levels of Ile and Val metabolic enzymes. The fact that similar phenotypes were induced in wild-type E. coli by the exogenous addition of 2-ketobutyrate and 2-aminobutyrate indicates that the 2 compounds contribute to the ΔyggS phenotypes. We showed that the initial cause of the keto acid imbalance was the reduced availability of coenzyme A (CoA); supplementation with pantothenate, which is a CoA precursor, fully reversed phenotypes conferred by the yggS mutation. The plasmid-borne expression of YggS and orthologs from Bacillus subtilis, Saccharomyces cerevisiae, and humans fully rescued the ΔyggS phenotypes. Expression of a mutant YggS lacking PLP-binding ability, however, did not reverse the ΔyggS phenotypes. These results demonstrate for the first time that YggS controls Ile and Val metabolism by modulating 2-ketobutyrate and CoA availability. Its function depends on PLP, and it is highly conserved in a wide range species, from bacteria to humans. 相似文献
29.
Kentaro Takai Yasunao Inoue Yasuko Konishi Atsushi Suwa Yoshiharu Uruno Harumi Matsuda Tomokazu Nakako Mutsuko Sakai Hiroyuki Nishikawa Gakuji Hashimoto Takeshi Enomoto Atsushi Kitamura Yasuaki Uematsu Akihiko Kiyoshi Takaaki Sumiyoshi 《Bioorganic & medicinal chemistry letters》2013,23(16):4644-4647
We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M1 and M4 agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats. 相似文献