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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.
Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects 总被引:8,自引:0,他引:8
S Harada K Sakamoto J K Seeley T Lindsten T Bechtold J Yetz G Rogers G Pearson D T Purtilo 《Journal of immunology (Baltimore, Md. : 1950)》1982,129(6):2532-2535
Eleven males with XLP were evaluated for EBV-specific antibodies during periods of 2 to 7 yr. Variable responses to EBV-specific antigens were found. All 11 patients had subnormal anti-EBNA titers, which probably reflected a T cell deficiency. The patients showed four different patterns in their anti-VCA response: 1) two boys who had experienced malignant lymphoma mounted no antibodies at all; 2) two patients showed intermittent anti-VCA titers; 3) four males had persistently elevated anti-VCA titers; and 4) three patients showed normal anti-VCA titers. ADCC against EBV-infected cells was abnormally low in six patients and was elevated in two patients given gamma-globulin. ADCC titers did not correlate with anti-VCA titers. However, most patients with XLP failed to effect regression of autologous EBV-infected lymphoblastoid cell lines, indicating a deficiency in long-lived T cell-mediated immunity to EBV. 相似文献
3.
S Ikei M Ogawa T Beppu C Ohara K Sakamoto H Sameshima H Arakawa Y Yamaguchi T Yamanaka S Kudo 《Cytokine》1992,4(6):581-584
In an attempt to investigate the interaction between the changes of cytokines and acute phase reactants after transcatheter arterial chemoembolization therapy (TACE), the levels of interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein (CRP) and pancreatic secretory trypsin inhibitor (PSTI) in the blood of patients with unresectable hepatocellular carcinoma (HCC) were measured. Before the therapy, serum IL-6 and plasma IL-8 levels were detectable in 77.8% and 28.5%, respectively, of patients with HCC. Levels of serum IL-6 and plasma IL-8 increased after TACE and reached a peak on day 3 in all patients (18/18) and in 87.5% of patients (12/14), respectively. Both blood levels of IL-6 and IL-8 reached a peak earlier than those of CRP and PSTI did after the therapy. When the maximal values of IL-6 were compared with those of CRP and PSTI, there were significant positive correlations (r = 0.63, P < 0.01 and r = 0.81, P < 0.01, respectively). Similarly, comparisons of the maximal values of IL-8 with those of CRP and PSTI gave a significant correlation (r = 0.68, P < 0.01 and r = 0.67, P < 0.05, respectively). However, no significant correlation was found between the elevation of IL-6 and IL-8. 相似文献
4.
Cs. Leranth H. Sakamoto N. J. MacLusky M. Shanabrough F. Naftolin 《Histochemistry and cell biology》1985,82(2):165-168
Summary A pre-embedding immunostaining procedure was developed using ferritin and peroxidase to enable simultaneous electron microscopic localization of two antigens in the same tissue section. This method was used to study the anatomic relationship between glutamic acid decarboxylase (GAD) immunoreactive axons and tyrosine hydroxylase (TH) — containing neurons of the rat arcuate nucleus. The findings provide ultrastructural evidence that GAD-immunoreactive terminals establish symmetric (Gray II) synapses on TH-reactive neurons. 相似文献
5.
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
6.
7.
Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level. 总被引:4,自引:0,他引:4
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The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2. 相似文献
8.
In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract 总被引:6,自引:0,他引:6
An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction. 相似文献
9.
Two tubificid species Limnodrilus hoffmeisteri and L. claparedeianus formed more than 93% of the total number of oligochaetes in the profundal. Limnodrilus spp. worms were found down to 33 cm in the sediment but in great numbers in the upper zone in June and October. Worms confined to the top 15 cm of sediment accounted for 53-92% of the total number. There were two annual maxima in population density and biomass, one in late spring (66000 inds m−2 , 17 g wet wt m−2 ) and the other in mid autumn (97000 inds m−2 , 176 g wet wt m−2 ). Two regression lines describing the effect of temperature on faecal production rate were obtained; Log F = 0.0604 T (°C) −0.7660 (below 15°C), Log F = 0.0266 T – 0.2170 (above 15°C). In total 26.8 kg dry wt m−2 of sediment was defecated annually by Limnodrilus spp. The sediment in the 0–10 cm stratum may pass through the guts of the worms 2.3 times a year. Sedimentation rates in profundal region were very low with respect to the faecal production rates of the tubificids. 相似文献
10.
H Sakamoto N J Maclusky P E Schwartz F Naftolin D Phil K Den S Takagi 《Endocrinologia japonica》1987,34(2):179-187
A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously. 相似文献