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T Kawada W S Shin Y Nakatsuru T Koizumi A Sakamoto T Nakajima Y Okai-Matsuo M Nakazawa H Sato T Ishikawa T Toyo-Oka 《Biochemical and biophysical research communications》1999,259(2):408-413
Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart. 相似文献
13.
S Masumura M Hashimoto Y Hashimoto T Sato I Kihara Y Watanabe 《European journal of applied physiology and occupational physiology》1982,48(2):157-161
Activities of glycolytic enzymes in the aorta were investigated in female Wistar rats. There were two groups of rats; one served as the control (sedentary rats), while the other group was forced to run on a treadmill for 10 weeks. In the control animals, the activities of hexokinase, phosphofructokinase and aldolase were relatively lower than those of the other glycolytic enzymes (phosphoglucose isomerase, lactate dehydrogenase and pyruvate kinase). After exercise, the activity of phosphofructokinase increased by 15%, whereas the other enzymatic activities were much the same as in the controls. Within the limits of the experiments, the increased percentage of phosphofructokinase was statistically significant (p less than 0.05). Since phosphofructokinase is a putative rate limiting enzyme, this enzymatic activation may indicate that glycolytic activity in the rat aorta is enhanced during and after running exercise. 相似文献
14.
The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity. 相似文献
15.
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
16.
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
17.
Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits. 相似文献
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Norio Masui Yumie Takagi Tetsu Nishikawa Makoto Yanabe Masato Nose Katsunori Sato 《Experimental Animals》2002,51(5):501-503
A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation. 相似文献