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991.
Translation inhibition reveals interaction of 2′-deoxy and 2′-O-methyl molecular beacons with mRNA targets in living cells
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Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo. Here, we demonstrate that starvation of cells and translation inhibition by blocking the mTOR or PI-3 kinase pathway could significantly reduce the fluorescence signal from 2′-deoxy molecular beacons (MBs) targeting K-ras and GAPDH mRNAs in living cells. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. We also found that, in targeting K-ras and GAPDH mRNAs, the signal level from MBs with 2′-O-methyl backbone did not change when translation was repressed. Taken together, our findings suggest that MBs with DNA backbone hybridize preferentially with mRNAs in their translational state in living cells, whereas those with 2′-O-methyl chemistry tend to hybridize to mRNA targets in both translational and nontranslational states. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology. 相似文献
992.
Diazotrophic endophytes of native black cottonwood and willow 总被引:1,自引:0,他引:1
Sharon L. Doty Brian Oakley Gang Xin Jun Won Kang Glenda Singleton Zareen Khan Azra Vajzovic James T. Staley 《Symbiosis (Philadelphia, Pa.)》2009,47(1):23-33
Poplar and willow are economically-important, fast-growing tree species with the ability to colonize nutrient-poor environments.
To initiate a study on the possible contribution of endophytes to this ability, we isolated bacteria from within surface-sterilized
stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis) in a riparian system in western Washington state. Several of the isolates grew well in nitrogen-limited medium. The presence
ofnifH, a gene encoding one of the subunits of nitrogenase, was confirmed in several of the isolates including species ofBurkholderia, Rahnella, Sphingomonas, andAcinetobacter. Nitrogenase activity (as measured by the acetylene reduction assay) was also confirmed in some of the isolates. The presence
of these diazotrophic microorganisms may help explain the ability of these pioneering tree species to grow under nitrogen
limitation. 相似文献
993.
Bulat I. Khayrutdinov Won Jin Bae Young Mi Yun Jie Hye Lee Takashi Tsuyama Jung Joo Kim Eunha Hwang Kyoung‐Seok Ryu Hae‐Kap Cheong Chaejoon Cheong Jung‐Soon Ko Takemi Enomoto P. Andrew Karplus Peter Güntert Shusuke Tada Young Ho Jeon Yunje Cho 《Protein science : a publication of the Protein Society》2009,18(11):2252-2264
In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C. 相似文献
994.
995.
Man‐IL Huh Yeoun‐Hee Kim Jong‐Hyuck Park Sung‐Won Bae Min‐Hee Kim Yongmin Chang Song‐Ja Kim Sun‐Ryung Lee Young‐Sup Lee Eun‐Jung Jin Jong‐Kyung Sonn Shin‐Sung Kang Jae‐Chang Jung 《Journal of cellular biochemistry》2009,108(2):476-488
In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
996.
997.
998.
Seok Won Hong In Seop Chang Yong Su Choi Tai Hak Chung 《Bioresource technology》2009,100(12):3029-3035
The aim of this study was to evaluate limiting factors affecting electricity output from sediment microbial fuel cells (sediment MFCs). In laboratory tests, various factors likely to be encountered in field application were divided into controllable and uncontrollable ones. Based on the findings, it could be suggested that the sediment MFCs can be operated with an anode to cathode area ratio of at least 5:1 and at high external loads (1000 Ω) when the cathode is closely placed to the anode, though DO concentration at the cathode must be kept above 3 mg/l. Furthermore, no significant effect on current production over a prolonged period was observed within the sediment temperature range of 20–35 °C, but was negatively affected by lower temperatures (10 °C). These observations provide important factors with respect to the construction and operation of sediment MFCs at field sites, which will aid in maximizing electricity output. 相似文献
999.
1000.
Eun Sil Kim Hee Jin Lee Won‐Gi Bang In‐Geol Choi Kyoung Heon Kim 《Biotechnology and bioengineering》2009,102(5):1342-1353
Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc. 相似文献