cellular assay
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2022 ◽  
Vol 3 (1) ◽  
pp. 101078
Author(s):  
Charles S. Lay ◽  
Daniel A. Thomas ◽  
John P. Evans ◽  
Emma J. Jones ◽  
Kelly M. Gatfield ◽  
...  

2021 ◽  
pp. 131755
Author(s):  
Wenya Jiao ◽  
Si Mi ◽  
Yaxin Sang ◽  
Qiuxia Jin ◽  
Bimal Chitrakar ◽  
...  

Author(s):  
Jason Wong ◽  
William Martelly ◽  
Shalini Sharma

2021 ◽  
Author(s):  
EunJee Park

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.


2021 ◽  
Author(s):  
EunJee Park

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.


2020 ◽  
Author(s):  
Koudedja Coulibaly ◽  
Marion Thauvin ◽  
Adyn Melenbacher ◽  
Clara testard ◽  
Evangelia Trigoni ◽  
...  

In this manuscript, we describe the implementation of a combinatorial apporach to synthesize a library of copper complexes, associated with an activity-based screening to discover the first peptidyl di copper complex mimicking CAT redox chemistry. The selected dinuclear complex was studied in detail and characterized for its CAT activity i<i>n vitro</i> and in cells. Very interestingly, despite moderate intrinsic catalysis constant, this complex was efficacious in a cellular assay.<br>


2020 ◽  
Author(s):  
Koudedja Coulibaly ◽  
Marion Thauvin ◽  
Adyn Melenbacher ◽  
Clara testard ◽  
Evangelia Trigoni ◽  
...  

In this manuscript, we describe the implementation of a combinatorial apporach to synthesize a library of copper complexes, associated with an activity-based screening to discover the first peptidyl di copper complex mimicking CAT redox chemistry. The selected dinuclear complex was studied in detail and characterized for its CAT activity i<i>n vitro</i> and in cells. Very interestingly, despite moderate intrinsic catalysis constant, this complex was efficacious in a cellular assay.<br>


2020 ◽  
Vol 21 (17) ◽  
pp. 6341
Author(s):  
Maria Sendino ◽  
Miren Josu Omaetxebarria ◽  
Gorka Prieto ◽  
Jose Antonio Rodriguez

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.


2020 ◽  
Author(s):  
Maria Sendino ◽  
Miren Josu Omaetxebarria ◽  
Gorka Prieto ◽  
Jose Antonio Rodriguez

ABSTRACTThe nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the “NES-binding groove” of CRM1 has been studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harbouring human micropeptides.


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