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101.
Kentaro Nagamine Yoko Kuzuhara Tsugunori Notomi 《Biochemical and biophysical research communications》2002,290(4):1195-1198
Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays. 相似文献
102.
103.
104.
We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine
(iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase
(IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type
cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the
labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin
research, such as receptor–ligand assays and cell metabolism studies. 相似文献
105.
Development of catheter-type optical oxygen sensor and applications to bioinstrumentation 总被引:3,自引:0,他引:3
A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 μm in diameter and 100 μm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO2 values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle. 相似文献
106.
Inaba Y Mizukami K Hamada-Sato N Kobayashi T Imada C Watanabe E 《Biosensors & bioelectronics》2003,19(5):423-431
A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx). 相似文献
107.
Developmental Expression and Substrate Specificities of Alfalfa
Caffeic Acid 3-O-Methyltransferase and
Caffeoyl
Coenzyme A 3-O-Methyltransferase in
Relation to
Lignification 总被引:2,自引:0,他引:2 下载免费PDF全文
108.
Kazuki Nagasawa Jun Miyaki Yuka Kido Youichirou Higashi Kentaro Nishida Sadaki Fujimoto 《Life sciences》2009,84(23-24):825-831
AimsRecently, we demonstrated that cultured mouse astrocytes exhibited basal channel opening of P2X7 receptor (P2X7R) in the absence of any exogenous ligand, but the regulatory mechanism involved was not elucidated. Since our preliminary experiments suggested possible involvement of peroxisome proliferator-activated receptor (PPAR) γ in the regulation, we examined whether PPARγ regulated P2X7R basal channel opening in mouse astrocytes.Main methodsP2X7R channel opening was assessed as to the uptake of a marker dye, YO-PRO-1® (YP), in the presence or absence of agonists and antagonists for PPARγ under a fluorescence microscope. Expression of PPARγ was evaluated by Western blotting and immunocytochemistry.Key findingsNSAIDs such as flufenamic acid (FFA) and indomethacin, which are a cyclooxygenase inhibitor and a PPARγ agonist, showed enhancing and inhibiting effects on YP uptake at low and high concentrations, respectively, and the enhanced uptake was abolished by periodate-oxidized ATP (oxATP), a selective P2X7R antagonist. The PPARγ agonists 15-deoxy-Δ12,14-prostaglandin J2 and ciglitazone decreased the basal and FFA-enhanced YP uptake, while the antagonist GW9662 increased YP uptake, this effect being blocked by the agonists and also by oxATP. PPARγ was distributed in the nucleus and cytosolic/membrane fraction of cultured mouse astrocytes.SignificanceThese findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPARγ. 相似文献
109.
110.
Tada-aki Kudo Hiroyasu Kanetaka Kentaro Mochizuki Kanako Tominami Shoko Nunome Genji Abe Hiroyuki Kosukegawa Toshihiko Abe Hitoshi Mori Kazumi Mori Toshiyuki Takagi Shin-ichi Izumi 《PloS one》2015,10(4)
To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine. 相似文献