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1.
2.
Jesus Torres-Bacete Prem Kumar Sinha Motoaki Sato Gaurav Patki Mou-Chieh Kao Akemi Matsuno-Yagi Takao Yagi 《The Journal of biological chemistry》2012,287(51):42763-42772
The bacterial H+-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (KArg-25, KArg-26, and KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed. 相似文献
3.
Jun Iwaki Kunio Kikuchi Yoshiaki Mizuguchi Yutaka Kawahigashi Hiroshi Yoshida Eiji Uchida Toshihiro Takizawa 《PloS one》2013,8(7)
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC. 相似文献
4.
Yasutaka Kakui Tomonari Sunaga Kunio Arai James Dodgson Liang Ji Attila Csikász-Nagy Rafael Carazo-Salas Masamitsu Sato 《Open biology》2015,5(6)
Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. 相似文献
5.
A respiration-deficient (RD) mutant was isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii. One strain among sixteen glycerol-non-utilizing mutants exhibited vigorous liberation of CO2 but no uptake of O2. Furthermore, this strain lacked cytochrome aa3 and had a reduced level of cytochrome b. The few mitochondria found in cells of this strain contained few or no cristae. Salt tolerance and intracellular accumulation of glycerol by the RD strain were almost equal to that of the wild-type strain in media containing NaCl up to 2.5 M. In media with more than 3 M NaCl, the growth of the RD mutant was retarded and the intracellular accumulation of glycerol was depressed in spite of ample production. 相似文献
6.
Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR 总被引:5,自引:0,他引:5
M Yazawa M Ikura K Hikichi L Ying K Yagi 《The Journal of biological chemistry》1987,262(23):10951-10954
Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function. 相似文献
7.
The changes of dominance rank among female Japanese monkeys of the Koshima group over a period of 29 years from 1957 were
studied. The dominance rank order was relatively stable in the early population growing phase, while large scale-changes of
dominance rank order occurred successively in the phase of population decrease brought about by the severe control of artificial
feeding after 1972. Nevertheless, the rank order of several females of the highest status was stable. Furthermore, the reproductive
success of these highest status females was high (Mori, 1979a;Watanabe et al., in prep.). Divergence of the dominance rank order fromKawamura's rules (Kawamura, 1958) was observed in the following respects: (1) Some females significantly elevated their rank depending on the leader
males. (2) If mothers died when their daughters were still juveniles or nulliparous, the dominance rank of some of these offspring
females was significantly lower than the mother's one. However 55% of daughters which lost their mothers at a young age inherited
the mother's rank. (3) Dominance among sisters whose mother had died when at least one of the daughters was under 6 years
old followed the rule of youngest ascendancy in 60% (Kawamura, 1958), and in 80% when both of the daughters were nulliparous at the mother's death.
The mean rate of aggressive interactions for each female with subordinates to her was calculated by dividing the total aggressive
interactions between the female in question and her subordinates by the number of subordinate females to the female in question.
A female which showed a high rate of aggressive interactions with her subordinates was categorized as an “Attacker”, and a
female showing a lower rate was categorized as a “Non-attacker”. Similarly, categories of “Attacked”, and “Non-attacked” were
distinguished by using the rate of aggressive interactions with dominant females. Several females which were once categorized
in one category in a year were repeatedly categorized in the same category over different years. The “Attacked” tended to
be females of higher rank, and “Non-attackers” tended to be females of lower rank. “The second-higher-status females”, were
“Attacked”, and their rank was unstable. In particular, females of lower rank within the lineage of the highest rank suffered
this kind of severe status. Most of the daughters of these females showed a sharp drop of rank, and died when they were still
at a young age, i.e. “the second-higher-status females” displayed low fitness. “Non-attackers” were significantly “Non-attacked”;
i.e. they were females which showed a non-social attitude. Females which underwent a drop of rank tended to be “Non-attackers”.
The most important factor which determined the females' rank was the memory of their dominance relations under the influence
of their mother [dependent rank (Kawai, 1958)] in their early life during development. This finding corresponds well with the results in baboons obtained byWalter (1980); the target females of aggressive interactions by adolescent females were determined by the rank of the mothers when
these adolescent females were born. 相似文献
8.
Conformational change of bovine serum albumin by heat treatment 总被引:1,自引:0,他引:1
Kunio Takeda Akira Wada Kazuo Yamamoto Yoshiko Moriyama Koichiro Aoki 《Journal of Protein Chemistry》1989,8(5):653-659
The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0. 相似文献
9.
We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovibrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovibrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: -250 mV less than or equal to e41 less than or equal to -220 mV, -325 mV less than or equal to e2 less than or equal to -320 mV, -335 mV less than or equal to e3 less than or equal to -330 mV, -360 mV less than or equal to e4 less than or equal to -355 mV, -5 mV less than or equal to I12 less than or equal to 20 mV, -10 mV less than or equal to I13 less than or equal to 5 mV, -15 mV less than or equal to I23 less than or equal to -5 mV, -15 mV less than or equal to I24 less than or equal to -10 mV, -25 mV, less than or equal to I34 less than or equal to -15 mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements. 相似文献
10.
Noriaki Murakami Yoshiyuki Tanaka Kunio Takishima Yuzo Minobe Makoto Matsuoka Sei-Ichiro Kiyota Shin-ichi Hatanaka Katsuhiro Sakano 《Journal of plant research》1990,103(4):419-434
We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of
the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It
contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding
regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature
protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant
species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed
plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution
rate per year per site for RuBisCO SSu was calculated to be 0.81×10−9 assuming that the separation between pteridophytes and seed plants arose 380 million years ago. 相似文献