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Kinji Endo Yōko Umeyama Junko Nakajima Hiroyasu Kawai 《Bioscience, biotechnology, and biochemistry》2013,77(7):1545-1551
Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA. 相似文献
13.
Susumu Hara Shigeki Arawaka Hiroyasu Sato Youhei Machiya Can Cui Asuka Sasaki Shingo Koyama Takeo Kato 《Molecular biology of the cell》2013,24(11):1649-1660
Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2. 相似文献
14.
Yasuhiro Suzuki Chandra Nath Roy Warunya Promjunyakul Hiroyasu Hatakeyama Kohsuke Gonda Junji Imamura Biju Vasudevanpillai Noriaki Ohuchi Makoto Kanzaki Hideo Higuchi Mitsuo Kaku 《Molecular and cellular biology》2013,33(15):3036-3049
The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines. 相似文献
15.
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
16.
Tetsuo Fukuzumi Hiroyasu Takahara Hajime Kaneko 《Bioscience, biotechnology, and biochemistry》2013,77(5):513-514
A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. Where Et1 and Et2 mean the optical density of the ethanol solution at 310 mμ prepared after t1 and t2 minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid. 相似文献
17.
Hiroyasu Koyama Katsura Kogure Kenji Mori Masanao Matsui 《Bioscience, biotechnology, and biochemistry》2013,77(4):915-920
Antimycinone A3, which is a neutral fragment of mild alkaline hydrolysate of antimycin A3, and its stereoisomers were synthesized stereoselectively from methyl trans-2-n-butylpent-3-enoate or methyl cis-2-n-butylpent-3-enoate, and natural antimycinone A3 was proved to possess Hα-Hβ and Hβ-Hγ trans configuration. 相似文献
18.
19.
Hiroyasu Kawai Tatsurokuro Tochikura 《Bioscience, biotechnology, and biochemistry》2013,77(10):1578-1593
Enzyme activities involved in the galactose metabolism of Torulopsis Candida grown on a. lactose medium were investigated with the cell-free extract and ammonium sulfate fraction. Remarkable activities of galactokinase, galactose-1-phosphate uridylyltransferase and UDPG pyrophosphorylase were detected, whereas UDPGal pyrophosphorylase activity was weak. UDPGal formation proceeded by the cell-free extract along a coupling reaction catalyzed by UDPG pyrophosphorylase and galactose-1-phosphate uridylyltransferase where UDPG or glucose-l-phosphate acted as a catalyst.The mechanism of UDPGal accumulation under the fermentative condition could be explained by a concerted inhibition of UDPGal-4- epimerase activity by 5′-UMP and galactose present as fermentation substrates. 相似文献
20.
A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases. 相似文献