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10.1177/0748730416668898 http://scihub22266oqcxt.onion/10.1177/0748730416668898 C5117186!5117186 !28112045     free     free     free Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 211.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Deprecated: Implicit conversion from float 245.2 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Warning: imagejpeg(C:\Inetpub\vhosts\kidney.de\httpdocs\phplern\28112045 .jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117       J+Biol+Rhythms 2016 ; 31 (6 ): 540-550 Nephropedia Template TP {{tp|p=28112045 |t=2016. In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells |pdf=|usr=}}{{28112045 }} gab.com Text In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells JO: J Biol Rhythms http://www.kidney.de/pget.php?&tw=1&pmid=28112045 #FOAvip Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (K(m)) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type. Twit Text FOAVip In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells ????J Biol Rhythms http://www.kidney.de/pget.php?&tw=1&pmid=28112045 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=28112045 #FOAvip English Wikipedia <ref>{{Cite pmid|28112045 }}</ref> In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells #MMPMID28112045 Feeney KA ; Putker M ; Brancaccio M ; O'Neill JS J Biol Rhythms 2016[Dec]; 31 (6 ): 540-550 PMID28112045 show ga Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (K(m)) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type. |Animals [MESH] |Cell Line [MESH] |Cell Line, Tumor [MESH] |Circadian Rhythm [MESH] |Fibroblasts/cytology/*metabolism [MESH] |Firefly Luciferin/metabolism [MESH] |Humans [MESH] |Kinetics [MESH] |Luciferases, Firefly/genetics/*metabolism [MESH] |Luminescent Measurements/methods [MESH] |Mice [MESH] |Period Circadian Proteins/genetics/*metabolism [MESH] |Suprachiasmatic Nucleus/metabolism [MESH]In-depth Characterization of Firefly Luciferase as a Reporter of Circa(epmc).pdf DeepDyve Pubget Overpricing