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51.
Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S‐phase transition 下载免费PDF全文
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Heidi LandmesserAnke Stein Bettina BlüschkeMelanie Brinkmann Sabine HunkeErwin Schneider 《生物化学与生物物理学报:生物膜》2002,1565(1):64-72
The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK2 complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a Vmax of 1.25 μmol Pi/min/mg and a Km of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIAGlc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes. 相似文献
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Ahn JS Musacchio A Mapelli M Ni J Scinto L Stein R Kosik KS Yeh LA 《Journal of biomolecular screening》2004,9(2):122-131
A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25. 相似文献
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Scerri TS Paracchini S Morris A MacPhie IL Talcott J Stein J Smith SD Pennington BF Olson RK DeFries JC Monaco AP Richardson AJ 《PloS one》2010,5(10):e13712
Background
Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis.Methodology/Principal Findings
Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L).Conclusions
Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required. 相似文献59.