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Chien-Jung Huang Laurent Schild Edward G. Moczydlowski 《The Journal of general physiology》2012,140(4):435-454
Voltage-gated Na+ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca2+ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca2+ or Na+ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca2+ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation. 相似文献
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In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution. 相似文献
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Marvin H. Caruthers Lincoln J. Mcbride Laurent P. Bracco John W. Dubendorff 《Nucleosides, nucleotides & nucleic acids》2013,32(1-2):95-105
Abstract An in situ method is described for synthesizing DNA which incorporates a new series of amidine protected deoxy-nucleosides and bis-dialkylaminophosphines as phosphitylating agents. These procedures were used to synthesize d(GGGAATTCCC) which was digested by EcoRI. 相似文献
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Laurent Chatel-Chaix Marie-Anne Germain Alena Motorina éric Bonneil Pierre Thibault Martin Baril Daniel Lamarre 《Journal of virology》2013,87(21):11704-11720
Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. 相似文献
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Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells 总被引:9,自引:0,他引:9
Annette Schmitt-Gräff Hans Pau Rolf Spahr Hans M. Piper Omar Skalli Giulio Gabbiani 《Differentiation; research in biological diversity》1990,43(2):115-122
Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed. 相似文献