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621.
Galietta LV Jayaraman S Verkman AS 《American journal of physiology. Cell physiology》2001,281(5):C1734-C1742
Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters. 相似文献
622.
Quinolinium based Cl- sensitive fluorescent indicators have been used extensively to measure intracellular Cl- activity. To define their fluorescence quenching mechanism, a series of N-methyl quinolinium derivatives were synthesized, including N-methylquinolinium (Q), 6-methylQ, 6-methoxyQ, 6-chloroQ, 3-bromoQ, 6-aminoQ and N-methylisoquinolinium. Stern-Volmer plots for quenching by Cl-, Br-, SCN-, I-, F-, OAc- and CO3(2-) from both intensity and lifetime measurements were linear. Bimolecular quenching rate constants (kq) decreased with increasing anion oxidation potentials and increased with increasing quinolinium reduction potentials. The free energy change for charge transfer (deltaG), calculated from indicator spectral and electrochemical properties, was found to correlate with log kq. These results suggest that quenching of quinolinium fluorescence in water by anions involves a charge-transfer quenching mechanism. Understanding the mechanism facilitates structure-based predictions of the anion sensitivities of quinolinium indicators to design improved Cl- indicators with tailored properties. 相似文献
623.
Samuel D Kumar TK Ganesh G Jayaraman G Yang PW Chang MM Trivedi VD Wang SL Hwang KC Chang DK Yu C 《Protein science : a publication of the Protein Society》2000,9(2):344-352
The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone. 相似文献
624.
Gregg W.C. Thomas Richard J. Wang Arthi Puri R. Alan Harris Muthuswamy Raveendran Daniel S.T. Hughes Shwetha C. Murali Lawrence E. Williams Harsha Doddapaneni Donna M. Muzny Richard A. Gibbs Christian R. Abee Mary R. Galinski Kim C. Worley Jeffrey Rogers Predrag Radivojac Matthew W. Hahn 《Current biology : CB》2018,28(19):3193-3197.e5
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627.
Shiny Abhijit Regin Bhaskaran Abirami Narayanasamy Anand Chakroborty Nagaraj Manickam Madhulika Dixit Viswanathan Mohan Muthuswamy Balasubramanyam 《Molecular and cellular biochemistry》2013,373(1-2):95-105
Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis. 相似文献
628.
Xiangjie Sun Akila Jayaraman Pavithra Maniprasad Rahul Raman Katherine V. Houser Claudia Pappas Hui Zeng Ram Sasisekharan Jacqueline M. Katz Terrence M. Tumpey 《Journal of virology》2013,87(15):8756-8766
The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity. 相似文献
629.
Lu Meng Farhad Forouhar David Thieker Zhongwei Gao Annapoorani Ramiah Heather Moniz Yong Xiang Jayaraman Seetharaman Sahand Milaninia Min Su Robert Bridger Lucas Veillon Parastoo Azadi Gregory Kornhaber Lance Wells Gaetano T. Montelione Robert J. Woods Liang Tong Kelley W. Moremen 《The Journal of biological chemistry》2013,288(48):34680-34698
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases. 相似文献
630.