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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.
Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors. 相似文献
3.
The developmental fate of Dictyostelium discoideum cells depends greatly on the cell-cycle position at the onset of starvation 总被引:4,自引:0,他引:4
The relationship between the development of Dictyostelium discoideum Ax-2 and the cell cycle at the onset of starvation was analysed with special reference to sorting behaviors during the formation of polarized cell masses (slugs), using a method for inducing good synchrony. Cells starved at different cell-cycle positions showed different developmental features during further culture. For example, cells just before mitosis and dividing cells were sorted out into the anterior prestalk zone of migrating slugs, while cells starved during most of the G2-phase, into the posterior prespore zone. Time courses of cell aggregation and tip formation were also found to vary greatly in a cell-cycle-related manner, and cells starved during the late G2-phase showed the most rapid development. Differential chemotaxis and cohesiveness are generally considered to be important for cell sorting in Dictyostelium development. In fact, remarkable differences in the chemotactic ability to a chemoattractant, cAMP, were detected among cells starved at any particular phase of the cell cycle. EDTA-resistant cohesiveness was also acquired differently depending on the cell cycle, and it was stronger in the cells showing more rapid aggregation. These findings indicate a close relation of the cell cycle to the cell sorting and pattern formation. The possible significance of the cell-cycle-related events presented here is discussed, with special emphasis on the process of cell aggregation. 相似文献
4.
cAMP in Anabaena cylindrica: Rapid Changes in Cellular Levels in Response to Changes in Extracellular Environments 总被引:2,自引:0,他引:2
The cellular level of cyclic 3',5'-AMP (cAMP) in the cyanobacteriumAnabaena cylindrica changed transiently in response to changesin extracellular environments. When the cells were transferedfrom dark to light, or anaerobic to aerobic conditions in thedark, the cAMP level rapidly decreased within one min and thengradually recovered. Addition of carbonyl cyanide m-chlorophenylhydrazone(CCCP) which inhibits ATP synthesis caused an increase in cAMPlevel in the light but not in the dark. The level of cAMP increasedseveral fold by lowering the pH from 8 to 6. On the contrary,a rise of pH from 6 to 8 caused a decrease in the cAMP level.It is suggested that the change in membrane electrochemicalpotential is involved in the regulation of cellular cAMP concentration. (Received January 27, 1989; Accepted June 19, 1989) 相似文献
5.
Masafumi Abe Naoya Nakamura Shirou Fukuhara Takamasa Hayashi Keiki Kawakami Kenkichi Kita Toshifumi Kinoshita Toyoro Ohsato Haruki Wakasa 《Virchows Archiv. B, Cell pathology including molecular pathology》1990,59(1):107-113
A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient
with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin
(SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and
antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both
the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common
ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line
T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings
indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage. 相似文献
6.
7.
Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts. 相似文献
8.
Nucleotide sequence and gene organization of ColE1 DNA 总被引:48,自引:0,他引:48
The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein). 相似文献
9.
Structure and antitumor activity of an alkali-soluble polysaccharide from Cordyceps ophioglossoides 总被引:3,自引:0,他引:3
H Yamada N Kawaguchi T Ohmori Y Takeshita S Taneya T Miyazaki 《Carbohydrate research》1984,125(1):107-115
A water-insoluble extracellular glucan (CO-1) was isolated from the precipitate formed on incubation of the culture filtrate of Cordyceps ophioglossoides at 37 degrees for 19 h. CO-1 was homogeneous as judged by h.p.l.c., electrophoresis, and ultracentrifugation, and the average molecular weight was determined by h.p.l.c. to be 632,000. The 1H- and 13C-n.m.r. and the i.r. spectra indicated that the glucosidic linkages in CO-1 were beta. From the results of methylation analysis, Smith degradation, n.m.r. studies, and enzymic hydrolysis, it was concluded that CO-1 is composed of a backbone of (1----3)-linked beta-D-glucopyranosyl residues with a beta-D-glucopyranosyl group attached to O-6 of every second D-glucopyranosyl residue of the backbone. CO-1 strongly inhibited the growth of Sarcoma 180 solid-type tumor. CO-1 polyalcohol, which was prepared by Smith degradation of CO-1, exhibited more-potent antitumor activity than CO-1. The absorption maximum of Congo Red shifted significantly in the presence of CO-1. 相似文献
10.
Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by 1H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters. 相似文献