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981.
Kensuke Ikeda Osamu Hoshi Hisashi Haga 《Biochemical and biophysical research communications》2010,400(1):181-186
The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (∼1 mm) into chromosomes (∼1 μm). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) IIα, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes. 相似文献
982.
Kensuke Kataoka 《Journal of microbiological methods》2010,82(3):342-346
Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. 相似文献
983.
Shinji Wakuta Shigeki Hamada Hiroyuki Ito Hideyuki Matsuura Kensuke Nabeta Hirokazu Matsui 《Phytochemistry》2010,71(11-12):1280-1288
Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing β-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an ~4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS–PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the Km and Vmax values of TAG were 31.7 μM and 0.25 μkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific β-glucosidase hydrolyzing TAG, which releases the physiologically active TA. 相似文献
984.
Eisuke Chikayama Atsushi Kurotani Takanori Tanaka Takashi Yabuki Satoshi Miyazaki Shigeyuki Yokoyama Yutaka Kuroda 《BMC bioinformatics》2010,11(1):113
Background
Efficient dissection of large proteins into their structural domains is critical for high throughput proteome analysis. So far, no study has focused on mathematically modeling a protein dissection protocol in terms of a production system. Here, we report a mathematical model for empirically optimizing the cost of large-scale domain production in proteomics research. 相似文献985.
Shuichi Hirose Kiyonobu Yokota Yutaka Kuroda Hiroshi Wako Shigeru Endo Satoru Kanai Tamotsu Noguchi 《BMC structural biology》2010,10(1):20
Background
Structural flexibility is an important characteristic of proteins because it is often associated with their function. The movement of a polypeptide segment in a protein can be broken down into two types of motions: internal and external ones. The former is deformation of the segment itself, but the latter involves only rotational and translational motions as a rigid body. Normal Model Analysis (NMA) can derive these two motions, but its application remains limited because it necessitates the gathering of complete structural information. 相似文献986.
987.
Shishido T Muraoka M Ueda M Seno M Tanizawa K Kuroda S Fukuda H Kondo A 《Applied microbiology and biotechnology》2006,73(3):505-511
Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host–vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 μg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC. 相似文献
988.
Effective display of metallothionein tandem repeats on the bioadsorption of cadmium ion 总被引:3,自引:0,他引:3
To increase the level of adsorption of heavy metal ions in surface-engineered yeasts, a yeast metallothionein (YMT) was tandemly
fused and displayed by means of an α-agglutinin-based display system. The display of the YMT and its tandem repeats was examined
by immunofluorescent labeling. The adsorption and recovery of Cd2+ on the cell surface was increasingly enhanced with increasing number of tandem repeats. All Cd2+-binding sites in the YMT tandem repeats were suggested to be completely occupied. To investigate the relationship between
cell-surface adsorption and protection against heavy metal ion toxicity, the tolerance of these surface-engineered yeasts
to Cd2+ was examined by growing in Cd2+-containing liquid medium. The rate of growth was found to be dependent on the number of displayed tandem repeats of YMT.
These results suggest that the characteristics of surface-engineered yeasts as a bioadsorbent were dependent on the ability
of the displayed proteins to bind metal ions, and the adsorption of heavy metal ions on the cell surface plays a major role
in the ability of the cells to resist the toxic effects of metal ions. 相似文献
989.
Murakawa T Okajima T Kuroda S Nakamoto T Taki M Yamamoto Y Hayashi H Tanizawa K 《Biochemical and biophysical research communications》2006,342(2):414-423
A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate alpha-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the alpha-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics. 相似文献
990.
To investigate the roles that the community effect and entrainment function of cultured cardiomyocyte play in decreasing beating fluctuation and reestablishing synchronized beating, we developed a single-cell-based two-dimensional network culture assay to measure and compare the dynamics of beating rhythm synchronization of individual cells before and after they form networks. Studying the formation of two-cell networks, we found that their synchronized beating tended to be determined by the cardiomyocyte whose beat rate fluctuated less than that of the other cardiomyocyte. We further found that the strength of this tendency increased with the number of cells in the network. These results indicate that (1) beating fluctuation is one of the important factors influencing the reestablishment of a stable synchronous beating rhythm, (2) the larger networks reduce fluctuation, and (3) the formation of a spatial network can itself stabilize cardiomyocyte beat rates. 相似文献