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1.
Tomohiro Hamasaki Takahiro Matsumoto Naoya Sakamoto Akiko Shimahara Shiori Kato Ayumi Yoshitake Ayumi Utsunomiya Hisayoshi Yurimoto Esteban C. Gabazza Tadaaki Ohgi 《Nucleic acids research》2013,41(12):e126
Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of 18O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging 18O atom into the phosphate group during the oxidation step of the synthetic cycle by using 18O water as the oxygen donor. The 18O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The 18O/16O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of 18O-labeled RNA, and this technique was used to determine the blood concentration of 18O-labeled RNA after administration to mice. 18O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies. 相似文献
2.
T Kanazawa 《The Journal of biological chemistry》1975,250(1):113-119
A large fraction of the Ca-2plus- and Mg-2plus-dependent ATPase (EC 3.6.1.3) in sarcoplasmic reticulum membranes solubilized with Triton X-100 was phosphorylated with Pi. The phosphorylation required Mg-2plus but was strongly inhibited by low concentrations of Ca-2plus. A Ca-2plus ion concentration of 30 muM caused half-maximum inhibition in the presence of 50 mM MgCl2. The phosphorylated enzyme showed a rapid turnover and was in dynamic equilibrium with Pi in the medium. At equilibrium the amount of the phosphorylated enzyme increased markedly with increased in the reaction temperature. The apparent standard free energy change, the apparent standard enthalpy change, and the apparent standard entropy change in the formation of the phosphorylated enzyme from the enzyme-phosphate complex in the presence of excess Mg-2plus at 37 degrees and pH 7.0 were, respectively, 0.35 Cal per mol, 15.9 Cal per mol, and 50.2 e.u. per mol. The susceptibility of the acid-denatured phosphorylated enzyme to hydroxylamine showed that the phosphorylated enzyme is of an acyl phosphate type. The present results are consistent with the probability that the phosphorylation results from reversal of late steps in the Ca-2plus transport process. The results clearly show that the phosphorylated enzyme is stabilized by a great increase in entropy upon its formation from the enzyme-phosphate complex. 相似文献
3.
Neutrophils become refractory to phorbol myristate acetate when treated with Ca2+-ionophore and Ca2+
M Nakagawara K Takeshige S Senju J Yoshitake S Minakami 《Journal of biochemistry》1987,101(3):601-608
Human neutrophils deprived of divalent cations by treatment with ionophore A23187 in the presence of ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed superoxide release when they were preincubated with calcium and then treated with the ionophore. The release was not observed when the ionophore was added first and then calcium was added more than 5 min later. The absence of the release in this case can be ascribed to a refractoriness of the cells to stimuli, because the cells did not release superoxide on stimulation with phorbol myristate acetate (PMA). The cells pretreated with either calcium or the ionophore alone did release superoxide on addition of PMA. The refractoriness of the cells to PMA depended on the concentrations of calcium and the ionophore and on the time interval between the two treatments. Calcium could be replaced with Cd2+ but not with Mg2+, Ba2+, or Sr2+. The release of granular enzymes was observed when the depleted cells were pretreated with the ionophore and then with calcium. These observations indicate that calcium has dual effects on the superoxide release of neutrophils, i.e., it stimulates the cells and makes them refractory to stimuli, depending on the time interval after the addition of the ionophore, and it also regulates the enzyme release by a different mechanism. 相似文献
4.
H Suzuki M Obara H Kuwayama T Kanazawa 《The Journal of biological chemistry》1987,262(32):15448-15456
Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes. 相似文献
5.
Insulin induces chloroquine-sensitive recycling of insulin-like growth factor II receptors but not of glucose transporters in rat adipocytes 总被引:1,自引:0,他引:1
Incubation of insulin-treated rat adipocytes with chloroquine, in a time- and concentration-dependent manner, was observed to inhibit the insulin-stimulated increase in insulin-like growth factor II (IGF-II) binding activity, whereas no significant change in IGF-II binding was observed in the absence of insulin. The incremental increase of insulin-stimulated IGF-II binding was inhibited 50% by 0.2 mM chloroquine within 15 min and was nearly completely abolished by 60 min. Interestingly, IGF-II binding was never observed to decrease below the binding value in cells without insulin treatment even when incubation was extended to 180 min. Scatchard analysis of IGF-II binding as well as the specific binding of an anti-IGF-II receptor antibody demonstrated that the loss of IGF-II binding in the insulin-stimulated chloroquine-treated adipocytes was due to a decrease in the number of cell-surface IGF-II receptors, whereas the total number of cellular IGF-II receptors was unaltered. The effect of chloroquine was observed to be reversible, temperature-dependent, and sensitive to the metabolic poison KCN. Furthermore, NH4Cl was also observed to inhibit insulin-stimulated increase in IGF-II binding. In contrast, chloroquine or NH4Cl did not inhibit the basal or insulin-stimulated glucose transport activity. Photoaffinity labeling of the glucose transporter with [3H]cytochalasin B also demonstrated that the basal and insulin-stimulated subcellular distribution of the glucose transporters was unaltered by chloroquine treatment. These results suggest that 1) insulin induces a constitutive, acidotropic agent-sensitive recycling of IGF-II receptor and 2) the glucose transporter and IGF-II receptor do not share the same insulin-regulated intracellular trafficking pathways. 相似文献
6.
Transfer of granulomatous inflammation with nonviable preparations of schistosome granulomas in naive mice 总被引:1,自引:0,他引:1
Hepatic granulomas of euthymic (nu/+) mice infected with Schistosoma mansoni were freeze-dried or freeze-thawed 3 times and transplanted subcutaneously into naive nu/+ and athymic (nu/nu) mice. The grafted sites, studied histologically, showed formation of organized granulomas in nu/+ mice similar to donor granulomas as observed after grafting of freshly isolated granulomas. On the other hand, in nu/nu mice, the nonviable transplants elicited small and disorganized granulomas, like hepatic granulomas in nu/nu mice with schistosomiasis, but different from fresh nu/+ transplants in nu/nu skin. The findings indicate viable cells are not required for transfer of granulomatous reactions, but T cells are needed for full expression. 相似文献
7.
Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium 总被引:3,自引:0,他引:3
A431 cells grew in protein-free Coon's modified Ham's F12 medium at a similar rate to that in medium supplemented with calf serum and secreted a growth factor capable of stimulating DNA synthesis in BALB/c3T3 cells. This factor had strong affinity for heparin and was partially purified from the conditioned medium by heparin-Sepharose affinity chromatography and molecular sieving on Bio-Gel P-60. The apparent molecular weight of the factor was 20-30K. Its activity was inhibited by heparin at concentrations of above 0.03 microgram/ml. 相似文献
8.
Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain 总被引:4,自引:0,他引:4
O Shinmi S Kimura T Sawamura Y Sugita T Yoshizawa Y Uchiyama M Yanagisawa K Goto T Masaki I Kanazawa 《Biochemical and biophysical research communications》1989,164(1):587-593
The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system. 相似文献
9.
Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination 总被引:2,自引:0,他引:2
O Shinmi S Kimura T Yoshizawa T Sawamura Y Uchiyama Y Sugita I Kanazawa M Yanagisawa K Goto T Masaki 《Biochemical and biophysical research communications》1989,162(1):340-346
We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system. 相似文献
10.
Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families 总被引:1,自引:0,他引:1
Ichiro Kanazawa Ikuko Kondo Joh-E Ikeda Teruaki Ikeda Yuichior Shizu Mitsuo Yoshida Hirotaro Narabayashi Shigetoshi Kuroda Hisayuki Tsunoda Eiji Mizuta Yoko Okuno Kiyotaka Sugawara Miho Murata Mafuyu Takahashi James F. Gusella 《Human genetics》1990,85(3):257-260
Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan. 相似文献