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1.
Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.  相似文献
2.
Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.  相似文献
3.
To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.  相似文献
4.
Mild stresses such as high temperature (30 degrees C) or a low H2O2 concentration induced transient cell cycle arrest at G1/S or G2/M depending on the cell cycle stage at which the stress was applied. When stresses were introduced during G0 or G1, the G1/S checkpoint was mainly used; when stresses were introduced after S phase, G2/M was the primary checkpoint. The slowing of cell cycle progression was associated with transient delays in expression of A-, B-, and D-type cyclins. The delay in expression of NtcycA13, one of the A-type cyclins, was most pronounced. The levels of expression of Ntcyc29 (a cyclin B gene) and of CycD3-1 differed most depending on the applied stress, suggesting that different cellular adjustments to mild heat and a low concentration of H2O2 are reflected in the expression of these two cyclins.  相似文献
5.
Kang HT  Hwang ES 《Life sciences》2006,78(12):1392-1399
2-Deoxyglucose (2-DG), a non-metabolizable glucose analogue, blocks glycolysis and inhibits protein glycosylation. It has been tested in multiple studies for possible application as an anticancer or antiviral therapeutic. The inhibitory effect of 2-DG on ATP generation made it a good candidate molecule as a calorie restriction mimetic as well. Furthermore, 2-DG has been utilized in numerous studies to simulate a condition of glucose starvation. Because 2-DG disrupts glucose metabolism, protein glycosylation, and ER quality control at the same time, a cellular or pathologic outcome could be easily misinterpreted without clear understanding of 2-DG's effect on each of these aspects. However, the effect of 2-DG on protein glycosylation has rarely been investigated. A recent study suggested that 2-DG causes hyperGlcNAcylation of proteins, while low glucose supply causes hypoGlcNAcylation. In certain aspects of cellular physiology, this difference could be disregarded, but in others, this may possibly cause totally different outcomes.  相似文献
6.
The whole mitochondrial genome (14,915 nt) of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia, Thoracica) was sequenced and characterized. It is the shortest of the 31 completely sequenced crustacean mitochondrial genomes, with the exception of a copepod Tigriopus japonicus (14,628 nt). It consists of the usual 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 relatively short non-coding region (294 nt). The thoracican cirripeds apart from Megabalanus volcano have the same arrangement of protein-coding genes as Limulus polypemus, but there are frequent tRNA gene translocations (at least 8). Some interesting translocation features that may be specific to the thoracican cirriped lineage are as follows: 1) trnK-trnQ lies between the control region and trnI, 2) trnA-trnE lies between trnN and trnS1, 3) trnP lies between ND4L and trnT, and 4) trnY-trnC lies between trnS2 and ND1. In P. mitella there are two trnL genes (L1 and L2) in the typical crustacean positions (ND1-L1-LrRNA and CO1-L2-CO2). The present result is compared and discussed with the other three cirriped mitochondrial genomes from one pedunculate (Pollicipes polymerus) and two sessiles (Tetraclita japonica and M. volcano) published so far. Mitochondrial protein phylogenies reconstructed by the BI and ML algorithms show that the thoracican Cirripedia is monophyletic (BPP 100/BP 100) and associated with Remipedia (BPP 98/BP 35). In addition, Oligostraca, including Ostracoda, Branchiura, and Pentastomida, is a monophyletic group (BPP 99/BP 68), and is basal to all the other examined arthropods. Remipedia + Cirripedia appears as an independent lineage within Arthropoda, apart from Thoracopoda (Malacostraca, Branchiopda, and Cephalocarida). The Thoracopoda is paraphyletic to Hexapoda. The present result suggests that the monophylies of Crustacea and Maxillopoda should be reconsidered.  相似文献
7.
Apio north-methanocarbocyclic nucleosides 1-3 with bicyclo[3. 1.0]hexane template were first synthesized. Introduction of hydroxymethyl substituent was efficiently and stereoselectively accomplished by aldol and retro-aldol reaction and fixed conformation was achieved from a modified Simmons-Smith cyclopropanation on a cyclopentane ring.  相似文献
8.
Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.  相似文献
9.
N-Ureido-quinoxalinedione derivatives have been discovered as leads for a novel series of dipeptidyl peptidase-IV (DPP-IV) inhibitors through high-throughput screening of our chemical library. A brief structure-activity relationship of the compounds was investigated. Among them, entry 5 showed the most potent inhibitory activity. The nitro group in quinoxaline moiety and the aromatic sulfonyl substituted ureido functional group seem to be important to increase the potency dramatically.  相似文献
10.
Poly(acrylic acid) (PAA)-patterned polystyrene (PS) substrates were prepared by ion beam lithography to control cell behaviors of mouse fibroblasts and human embryonic kidney cells. Thin PAA films spin-coated on non-biological PS substrates were selectively irradiated with energetic proton ions through a pattern mask. The irradiated substrates were developed with deionized water to generate negative-type PAA patterns. The surface characteristics of the resulting PAA-patterned PS surface, such as surface morphology, chemical structure and composition and wettability, were investigated. Well-defined 100 μm PAA patterns were effectively formed on relatively hydrophobic PS substrates by ion beam lithography at higher fluences than 5 × 1014 ions/cm2. Moreover, based on the in vitro cell culture test, cells were adhered and proliferated favorably onto hydrophilic PAA regions separated by hydrophobic PS regions on the PAA-patterned PS substrates, and thereby leading to the formation of well-defined cell patterns.  相似文献
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