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61.
Pham Huynh Ninh Kohsuke Honda Yukako Yokohigashi Kenji Okano Takeshi Omasa Hisao Ohtake 《Applied and environmental microbiology》2013,79(6):1996-2001
The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher. 相似文献
62.
Kazunori Ushimaru Smith Sangiambut Nicholas Thomson Easan Sivaniah Takeharu Tsuge 《Applied microbiology and biotechnology》2013,97(3):1175-1182
The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaCRe) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaCRe. In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279–6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaCRe was conducted in the presence of TritonX-100, and PhaCRe obeyed Michaelis–Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3′-dephospho CoA had no inhibitory effect. Furthermore, PhaCRe showed a considerably reduced reaction rate for 3′-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis–Menten kinetics. These results suggest that the 3′-phosphate group of CoA plays a critical role in substrate recognition by PhaCRe. 相似文献
63.
Shinichi Mochizuki Naho Kanegae Koichi Nishina Yumi Kamikawa Kazunori Koiwai Hiroyasu Masunaga Kazuo Sakurai 《生物化学与生物物理学报:生物膜》2013,1828(2):412-418
Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE = 1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency. 相似文献
64.
Keiju Okano Takeshi Nakamura Yuji Kamiya Susumu Ikegami 《Bioscience, biotechnology, and biochemistry》2013,77(3):805-807
Cultured crown gall cells of Catharanthus roseus Don (Vinca rosea L.) was found to contain brassinosteroids. These were identified as brassinolide and castasterone by GC/MS. This is the first conclusive identification of endogenous brassinosteroids in cultured cells. 相似文献
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68.
Kazunori Sasaki Tomoyuki Oki Yumi Kai Yoichi Nishiba 《Bioscience, biotechnology, and biochemistry》2013,77(8):1308-1314
The purpose of this study was to investigate the effect of repeated harvesting on the content of caffeic acid (CA) and seven species of caffeoylquinic acids (CQAs) in sweet potato leaves using a newly developed high-performance liquid chromatography method. Six cultivars and two breeding lines were used in this study. Leaves were collected at monthly intervals from 1st harvest (May) to 4th harvest (August) in 2011 and 2012. ANOVA analysis revealed that the contents of CQAs were significantly different among all cultivars and breeding lines, but no significant differences were found for CA. No annual variation was confirmed in CA and CQAs. Repeated harvest of sweet potato leaves affected the content of only 4-CQA and 5-CQA. Post-hoc comparisons using Tukey’s method indicated that the contents of 4-CQA and 5-CQA in sweet potato leaves harvested at first time were significantly higher compared to those at the other harvest times. 相似文献
69.
Akihito Tanaka Knut Woltjen Katsuya Miyake Akitsu Hotta Makoto Ikeya Takuya Yamamoto Tokiko Nishino Emi Shoji Atsuko Sehara-Fujisawa Yasuko Manabe Nobuharu Fujii Kazunori Hanaoka Takumi Era Satoshi Yamashita Ken-ichi Isobe En Kimura Hidetoshi Sakurai 《PloS one》2013,8(4)
The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. 相似文献
70.
Kazunori Ohkawara Marc‐Andre Cornier Wendy M. Kohrt Edward L Melanson 《Obesity (Silver Spring, Md.)》2013,21(2):336-343