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51.
Izumi Uno Takashi Kozaka Daisuke Miwa Yoji Kitamura Mohammad Anwar-ul Azim Kazuma Ogawa Junichi Taki Seigo Kinuya Kazuhiro Shiba 《PloS one》2016,11(1)
Purpose
To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.Procedures
Radioiodinated o-iodo-trans-decalinvesamicol ([125I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [125I]OIDV was separated into its two optical isomers (-)-[125I]OIDV and (+)-[125I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [125I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [125I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [125I]OIDV isomers and (-)-[123I]OIDV for SPECT at 60 min postinjection.Results
VAChT binding affinity (Ki) of (-)-[125I]OIDV and (+)-[125I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[125I]OIDV was the same as that of (+)-[125I]OIDV. However, (+)-[125I]OIDV clearance from the brain was faster than (-)-[125I]OIDV. At 30 min post-injection, accumulation of (-)-[125I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[125I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[125I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[125I]OIDV in these regions. Furthermore, (-)-[123I]OIDV for SPECT showed the same regional brain distribution as (-)-[125I]OIDV.Conclusion
These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain. 相似文献52.
Junichiro Futami Yuki Atago Akari Azuma Endy Widya Putranto Rie Kinoshita Hitoshi Murata Masakiyo Sakaguchi 《Biochemistry and Biophysics Reports》2016
It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems. 相似文献
53.
Yuki Miyamoto Tomohiro Torii Kazuko Kawahara Akito Tanoue Junji Yamauchi 《Biochemistry and Biophysics Reports》2016
During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins. 相似文献
54.
Yuki Sato Shiori Sato Takahiro Kikuchi Asumi Nonaka Yuki Kumagai Akira Sasaki Masayuki Kobayashi 《Analytical biochemistry》2016
Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. 相似文献
55.
Subramanya Rao Yuki Chan Donnabella C. Bugler-Lacap Ashish Bhatnagar Monica Bhatnagar Stephen B. Pointing 《Indian journal of microbiology》2016,56(1):35-45
A culture-independent diversity assessment of archaea, bacteria and fungi in the Thar Desert in India was made. Six locations in Ajmer, Jaisalmer, Jaipur and Jodhupur included semi-arid soils, arid soils, arid sand dunes, plus arid cryptoendolithic substrates. A real-time quantitative PCR approach revealed that bacteria dominated soils and cryptoendoliths, whilst fungi dominated sand dunes. The archaea formed a minor component of all communities. Comparison of rRNA-defined community structure revealed that substrate and climate rather than location were the most parsimonious predictors. Sequence-based identification of 1240 phylotypes revealed that most taxa were common desert microorganisms. Semi-arid soils were dominated by actinobacteria and alpha proteobacteria, arid soils by chloroflexi and alpha proteobacteria, sand dunes by ascomycete fungi and cryptoendoliths by cyanobacteria. Climatic variables that best explained this distribution were mean annual rainfall and maximum annual temperature. Substrate variables that contributed most to observed diversity patterns were conductivity, soluble salts, Ca2+ and pH. This represents an important addition to the inventory of desert microbiota, novel insight into the abiotic drivers of community assembly, and the first report of biodiversity in a monsoon desert system. 相似文献
56.
57.
Quantitative and antioxidative behavior of Trolox in rats’ blood and brain by HPLC‐UV and SMFIA‐CL methods 下载免费PDF全文
Mitsuhiro Wada Misato Wada Rie Ikeda Yuki Fuchigami Hironari Koyama Susumu Ohkawara Shigeru Kawakami Naotaka Kuroda Kenichiro Nakashima 《Luminescence》2016,31(2):414-418
Trolox, a water‐soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi‐microflow injection analysis, chemiluminescence detection and HPLC‐UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 103 antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0–360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood–brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
58.
Yuki Takayasu Yusuke Ogura Ryo Towada 《Bioscience, biotechnology, and biochemistry》2016,80(8):1459-1463
The first synthesis of the (9R,13R)-stereoisomer of LDS1, a flower-inducing oxylipin isolated from Lemna paucicostata, has been achieved from a known allylic alcohol by a seven-step sequence that involves the Horner–Wadsworth–Emmons olefination to construct its full carbon framework and an enzymatic hydrolysis of a penultimate methyl ester intermediate to provide the target molecule. 相似文献
59.
Murakami Shinsuke Nakatani Jun Nakajima Kenichi Amasawa Eri Ii Ryota Hayashi Kiyotada Yoshikawa Naoki Daigo Ichiro Kishita Yusuke Ihara Tomohiko Shobatake Koichi Kudoh Yuki Motoshita Masaharu Kanemoto Keiichiro Hara Minako Kashiwagi Aiichiro Hashimoto Seiji Shigetomi Yosuke Kanzaki Masayuki Kikuchi Yasunori Ohno Hajime Fukushima Yasuhiro 《The International Journal of Life Cycle Assessment》2019,24(8):1544-1552
The International Journal of Life Cycle Assessment - 相似文献
60.
Hiroyuki Ichida Ryouhei Morita Yuki Shirakawa Yoriko Hayashi Tomoko Abe 《The Plant journal : for cell and molecular biology》2019,98(2):301-314
Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations. 相似文献