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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
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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
4.
J-H Kim K W Park E-W Lee W-S Jang J Seo S Shin K-A Hwang J Song 《Cell death and differentiation》2014,21(4):594-603
The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ. 相似文献
5.
Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 hу to 0.12 hу. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g lу and 9 g lу, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g lу and 49 g lу, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture. 相似文献
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The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts. 相似文献
8.
The ultrastructural features of the biflagellate motile cells of six different species of the Chlorophyceae, namely Dunaliella lateralis (Polyblepharidaceae, Chlamydomonadales), Chlorococcum hypnosporum, Spongiochloris spongiosa, Protosiphon botryoides (Chlorococcaceae, Chlorococcales), Tetracystis aeria and Pseudotetracystis terrestris (Tetracystidaceae, Chlorococcales), were examined with an emphasis on the flagellar apparatus (FA). They have different vegetative characteristics, such as, being motile or nonmotile, variations in chloroplast morphology, possession of one or more nuclei, and reproductive features such as formation of tetrahedral tetrads, and naked or walled zoospores. Ultrastructural differences amongst reproductive cells of the six species include variations in cell surface structure, basal body to basal body angle, beamlike extensions of the distal fiber, extensive connections of the proximal sheath between basal bodies, two-membered rootlets, striated microtubule-associated components, two-membered rootlet-nucleus and/or mitochondria connections, X-membered rootlets, connections of rootlets and basal bodies, rhizoplasts and accessory basal bodies. All six species possess pyrenoids penetrated by thylakoid membranes, and the FA typical of the Chlorophyceae (sensu Mattox and Stewart, 1984). These six species should be divided into two groups. The first includes D. lateralis, C. hypnosporum, and T. aeria, in which accessory basal bodies are present, the basal body to basal body angle is relatively fixed, and a cell wall or surface coat is present. The second group includes Ps. terrestris, S. spongiosa, and Pr. botryoides, in which accessory basal bodies are absent, the basal body to basal body angle is variable and the zoospores are naked. 相似文献
9.
A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins. 相似文献
10.
Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C 总被引:2,自引:0,他引:2
We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis. 相似文献