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171.
Wei Choon Alvin Koh Eun Sang Choe Dong Kun Lee Seung-Cheol Chang Yoon-Bo Shim 《Biosensors & bioelectronics》2009,25(1):211-217
An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H+-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H+ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml−1. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis. 相似文献
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Innokentiy Maslennikov Martin Krupa Christopher Dickson Luis Esquivies Katherine Blain Georgia Kefala Senyon Choe Witek Kwiatkowski 《Journal of structural and functional genomics》2009,10(1):25-35
Abstract Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane
proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of
an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used
in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent
complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably,
this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one
particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein
oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties
through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for
solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact
protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating
PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore
more effective than centrifugal ultrafiltration devices. 相似文献
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176.
Elvira Valera Michael J. Isaacs Yasuhiko Kawakami Juan Carlos Izpisúa Belmonte Senyon Choe 《PloS one》2010,5(6)
Background
Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.Methodology/Principal Findings
We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.Conclusions/Significance
Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance. 相似文献177.
178.
The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo. 相似文献
179.
Sang-im Lee Kyungseon Seo Wonyoung Lee Woohjung Kim Jae Chun Choe Piotr Jabłoński 《Journal of Ethology》2011,29(3):401-407
We report the first case of non-parental infanticide in the Black-billed Magpie Pica pica. Using a video camera installed in the victims’ nest, we recorded repeated visits (over 4 days) of an adult (each time one
bird) who attacked six nestlings at each visit until they died or were evicted. The nest was one of 58 nests filmed over four
breeding seasons. Collected evidence suggests that the perpetrator(s) might have been the female breeder of the neighbouring
nest, possibly also her male partner. The parental female aggressively attacked the perpetrator. Post-infanticide expansion
of breeding territory by the suspected perpetrator is the hypothetical ultimate explanation of the observed infanticide. Movie
clips from inside the nest are shown at: , , , and . 相似文献
180.
Altitude influences forest structure and food abundance and distribution, which in turn affect primate feeding and ranging patterns. Javan gibbons (Hylobates moloch) are endemic to forests spanning a broad range of altitudes on Java, Indonesia. Most information about Javan gibbon behavior comes from studies in lowland forests, while the vast majority of wild gibbons remaining inhabit hill and lower montane forests. We studied the diets, activity patterns, and ranging behavior of three gibbon groups in hill/lower montane (950-1,100?m asl) forest in the Gunung Halimun-Salak National Park (GHSNP) from April 2008 to March 2009. The mean home range size was 37?ha and the mean daily path length was 1,180?m. The study groups spent 36% of time feeding, 41% resting, 15% traveling, 6% engaging in social behavior, and 2% in aggressive interactions. Fruit was the most important food (63% of feeding time) followed by leaves (24%), and flowers (12%). Our results suggest that Javan gibbons in higher elevation habitats have substantially larger home ranges than lowland populations, despite broad similarity in their activity budgets and diets. Conservation managers should consider the effects of altitude and habitat quality on gibbon ranging behavior when developing habitat corridors, selecting sites for translocation or reintroduction projects, and designating and managing protected areas. 相似文献