scholarly journals 306 Involvement of toll-like receptor 4 in PCSK9-mediated biological effects in cell culture models

2021 ◽  
Vol 23 (Supplement_G) ◽  
Author(s):  
Valentina Scalise ◽  
Chiara Sanguinetti ◽  
Michele Lai ◽  
Tommaso Neri ◽  
Alessandro Celi ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Cell Line ◽  
Reporter Gene ◽  
Specific Binding ◽  
Biological Effects ◽  
Negative Control ◽  
Hek293 Cells ◽  
Gene Assay ◽  
Culture Models ◽  
A Cell

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease was supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway was found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. Results hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P < 0.001] to an extent comparable to Lipopolysaccharide (LPS), the specific agonist of TLR4. Colocalization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P < 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behaviour that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFKB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

scholarly journals 305 Usefulness of CHA2DS2-VASc scoring system for predicting risk of embolism in patients with cardiac tumours: a single-centre study

2021 ◽  
Vol 23 (Supplement_G) ◽  
Author(s):  
Ilenia Magnani ◽  
Pasquale Paolisso ◽  
Chiara Chiti ◽  
Francesco Angeli ◽  
Alberto Foa ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Cell Line ◽  
Reporter Gene ◽  
Specific Binding ◽  
Negative Control ◽  
Hek293 Cells ◽  
Cardiac Tumours ◽  
Gene Assay ◽  
Single Centre Study ◽  
A Cell

Abstract Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease has been supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway has been found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods and results Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P < 0.001] to an extent comparable to lipopolysaccharide, the specific agonist of TLR4. Co-localization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P < 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behavior that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFkB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.

Glycine-extended gastrin regulates HEK cell growth

1999 ◽  
Vol 277 (2) ◽  
pp. R572-R581 ◽  
Author(s):  
Vinzenz M. Stepan ◽  
Dieter F. Krametter ◽  
Masashi Matsushima ◽  
Andrea Todisco ◽  
John Delvalle ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Cell Line ◽  
Thymidine Incorporation ◽  
Specific Binding ◽  
Biological Effects ◽  
Human Kidney ◽  
Hek 293 ◽  
High Concentrations

Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH2) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH2, glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH2 and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH2 can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH2 and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for 125I-labeled Leu15-G17-NH2and125I-Leu15-G2—17-Gly. Both G-NH2 and G-Gly induced a dose-dependent increase in [3H]thymidine incorporation, and both peptides together significantly increased [3H]thymidine incorporation above the level of either peptide alone. G-NH2 and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH2 and G-Gly lead to a significant reduction in [3H]thymidine incorporation. G-NH2 but not G-Gly increased intracellular Ca2+concentration. We conclude that G-NH2 and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.

417 REPORTER GENE BIOASSAY OF FOLLICLE-STIMULATING HORMONE ACTIVITY IN MARE SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 365
Author(s):  
F. Sahmi ◽  
K. Sayasith ◽  
V. Portela ◽  
C. A. Price
Keyword(s):  
Cell Line ◽  
Granulosa Cell ◽  
Cell Lines ◽  
Reporter Gene ◽  
Colorimetric Assay ◽  
Animal Health ◽  
Major Effect ◽  
Hek293 Cells ◽  
Time Interval ◽  
Dependent Manner

Equine chorionic gonadotropin is secreted by the mare placenta and possesses both LH and FSH bioactivities in nonequine species. In ruminants, eCG is used commercially to induce superovulation. The production of commercial eCG is hampered by the variation in FSH and LH bioactivity of eCG between mares, and potentially results in batch-to-batch variation in eCG bioactivity. The objective of this study was to establish a cell-line-based bioassay of FSH activity in serum for use at eCG production facilities. Several cell lines were used for this study: HEK293 (kidney cells), KGN (a human granulosa cell line), and a new bovine granulosa cell line. The HEK293 and bovine granulosa cell lines did not express the FSH receptor (FSHR); therefore, the strategy was to cotransfect those cells with a FSHR expression plasmid and a cAMP reporter gene (β-galactosidase; β-Gal). The KGN cells transfected with β-Gal failed to respond to FSH and were not used further. The HEK293 and bovine cell lines responded to FSH in a dose-dependent manner, with a visible increase in β-Gal activity measured by colorimetric assay. The cells responded to eCG but not to LH, IGF1, or estradiol, demonstrating specificity for FSH activity. The minimum time of incubation required for clear bioactivity was 4 h. Activity was detected in serum from pregnant but not estrous mares. Attempts to create stable cell lines expressing both FSHR and β-Gal plasmids were not productive. We therefore attempted to create frozen batches of transiently transfected HEK293 cells. Several incubation conditions were tested and we succeeded in detecting β-Gal activity in response to eCG in thawed cells. The choice of serum during transfection had a major effect on the ability of the cells to respond to eCG after thawing, and the time interval between transfection and freezing significantly altered the magnitude of the response to eCG. The cells responded visibly to eCG treatment after 4-h incubation. In summary, we have developed a reasonably fast, colorimetric bioassay for FSH activity that can be used for serum in an on-farm setting. Supported by NSERC, AAFC, and Bioniche Animal Health.

scholarly journals Selective Activation of Zebrafish Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell Line

2011 ◽  
Vol 125 (2) ◽  
pp. 439-449 ◽  
Author(s):  
Anne Cosnefroy ◽  
François Brion ◽  
Emmanuelle Maillot-Maréchal ◽  
Jean-Marc Porcher ◽  
Farzad Pakdel ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Cell Line ◽  
Reporter Gene ◽  
Liver Cell ◽  
Reporter Gene Assay ◽  
Receptor Subtypes ◽  
Selective Activation ◽  
Liver Cell Line ◽  
Gene Assay ◽  
Zebrafish Liver

scholarly journals A Cell-Based β-Lactamase Reporter Gene Assay for the CREB Signaling Pathway

2009 ◽  
Vol 3 ◽  
pp. 7-12 ◽  
Author(s):  
Menghang Xia ◽  
Vicky Guo ◽  
Ruili Huang ◽  
James Inglese ◽  
Marshall Nirenberg ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Gene Assay ◽  
A Cell

scholarly journals Validation of a cell-based colorimetric reporter gene assay for the evaluation of Type I Interferons

2019 ◽  
Vol 22 ◽  
pp. e00331 ◽  
Author(s):  
Ignacio Mejía-Calvo ◽  
Leslie Muñoz-García ◽  
Alexis Jiménez-Uribe ◽  
Rosa Camacho-Sandoval ◽  
Edith González-González ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Type I Interferons ◽  
Type I ◽  
Gene Assay ◽  
A Cell

A Cell-Based Reporter Gene Assay for Determining Induction of CYP3A4 in a High-Volume System

2002 ◽  
Vol 303 (1) ◽  
pp. 412-423 ◽  
Author(s):  
Judy Raucy ◽  
Lyndon Warfe ◽  
Mei-Fei Yueh ◽  
Scott W. Allen
Keyword(s):  
Reporter Gene ◽  
High Volume ◽  
Reporter Gene Assay ◽  
Gene Assay ◽  
A Cell

scholarly journals Development of a Profiling Strategy for Metabolic Mixtures by Combining Chromatography and Mass Spectrometry with Cell-Based GPCR Signaling

2012 ◽  
Vol 17 (10) ◽  
pp. 1329-1338 ◽  
Author(s):  
Saskia Nijmeijer ◽  
Henry F. Vischer ◽  
Anders F. Rudebeck ◽  
Frank Fleurbaaij ◽  
David Falck ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Reporter Gene ◽  
Metabolic Profiling ◽  
Reporter Gene Assay ◽  
Lead Compounds ◽  
Analytical Separations ◽  
Liquid Chromatographic Separation ◽  
Gene Assay ◽  
A Cell ◽  
Liquid Chromatographic

In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H4 receptor (hH4R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH4R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction–driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process.

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