scholarly journals Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

2012 ◽  
Vol 1817 (9) ◽  
pp. 1660-1670 ◽  
Author(s):  
Martin Jastroch ◽  
Verena Hirschberg ◽  
Martin Klingenspor
Keyword(s):  
Functional Characterization ◽  
Hek293 Cells ◽  
Proton Leak ◽  
Mitochondrial Uncoupling

scholarly journals Characterization of an N-terminal Nav1.5 channel variant – a potential risk factor for arrhythmias and sudden death?

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Stefanie Scheiper-Welling ◽  
Paolo Zuccolini ◽  
Oliver Rauh ◽  
Britt-Maria Beckmann ◽  
Christof Geisen ◽  
...  
Keyword(s):  
Sudden Death ◽  
Functional Characterization ◽  
Missense Variant ◽  
Hek293 Cells ◽  
Patch Clamp Technique ◽  
Gene Encoding ◽  
Heterozygous Variant ◽  
Cardiac Sodium Channel ◽  
Electrophysiological Measurements

Abstract Background Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. Methods Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. Results Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. Conclusion The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

scholarly journals Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells

2021 ◽  
Vol 15 ◽  
Author(s):  
Rebecca E. Roberts ◽  
Jothi Kumar Yuvaraj ◽  
Martin N. Andersson
Keyword(s):  
Bark Beetles ◽  
Codon Optimization ◽  
Odorant Receptor ◽  
Functional Characterization ◽  
Stable Expression ◽  
Hek293 Cells ◽  
Western Blots ◽  
Protein Levels ◽  
Hek Cells

Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization (“de-orphanization”) using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus (“Ityp”) and Dendroctonus ponderosae (“Dpon”), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(−)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes.

Functional Characterization of a Drosophila Mitochondrial Uncoupling Protein

2004 ◽  
Vol 36 (3) ◽  
pp. 219-228 ◽  
Author(s):  
Yih-Woei C. Fridell ◽  
Adolfo Sánchez-Blanco ◽  
Brian A. Silvia ◽  
Stephen L. Helfand
Keyword(s):  
Uncoupling Protein ◽  
Functional Characterization ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Uncoupling Protein

scholarly journals Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

2021 ◽  
Author(s):  
Christoph Gstöttner ◽  
Tao Zhang ◽  
Anja Resemann ◽  
Sophia Ruben ◽  
Stuart Pengelley ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Structural Features ◽  
Host Cells ◽  
Hek293 Cells ◽  
Functional Features

AbstractAs the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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