scholarly journals Angular-type Furocoumarins from the Roots of Angelica atropurpurea and their Inhibitory Activity on the NFAT Signal Transduction Pathway

2014 ◽  
Vol 9 (12) ◽  
pp. 1934578X1400901
Author(s):  
Azumi Nagasawa ◽  
Mitsuyoshi Sakasai ◽  
Daishi Sakaguchi ◽  
Shigeru Moriwaki ◽  
Yoshinori Nishizawa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Inhibitory Activity ◽  
Cultured Cells ◽  
Signal Transduction Pathway ◽  
Spectroscopic Studies ◽  
Luciferase Reporter ◽  
Transduction Pathway ◽  
Luciferase Reporter Gene ◽  
Activated T Cells ◽  
Gene Assay

One new (1) and two known angular-type (2,3) furocoumarins, isoarchangelicin (1), archangelicin (2), and 2′-angeloyl-3′-isovaleryl vaginate (3), were isolated from the roots of Angelica atropurpurea. The structure of the new compound was established on the basis of one- and two-dimensional NMR spectra and other spectroscopic studies. The inhibitory activity of these three compounds and a deacylated form of archangelicin (4) on the nuclear factor of activated T cells (NFAT) signal transduction pathway was tested by NFAT-responsive luciferase reporter gene assay in cultured cells. Although 4 did not exhibit inhibitory activity on NFAT signaling, 1–3 exhibited dose-dependent inhibition with IC50 values of 16.5 (1), 9.0 (2), and 9.2 (3) μM.

scholarly journals Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators

2000 ◽  
Vol 5 (4) ◽  
pp. 255-261 ◽  
Author(s):  
G. C. Terstappen ◽  
A. Giacometti ◽  
E. Ballini ◽  
L. Aldegheri
Keyword(s):  
Reporter Gene ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Signal Transduction Pathway ◽  
Resistant Cell ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Camp Levels

For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC50 of 0-9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.

scholarly journals Curcumin induces paraoxonase 1 in cultured hepatocytes in vitro but not in mouse liver in vivo

2010 ◽  
Vol 105 (2) ◽  
pp. 167-170 ◽  
Author(s):  
Charlotte Schrader ◽  
Christina Schiborr ◽  
Jan Frank ◽  
Gerald Rimbach
Keyword(s):  
Cultured Cells ◽  
Paraoxonase 1 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Huh7 Cells ◽  
Gene Assay ◽  
B6c3f1 Mice

Paraoxonase 1 (PON1) is an enzyme that is mainly synthesised in the liver and protects LDL from oxidation, thereby exhibiting antiatherogenic properties. Using a luciferase reporter gene assay, we tested curcumin for its ability to induce PON1 in Huh7 hepatocytes in culture. Curcumin ( ≥ 10 μmol/l) dose-dependently induced PON1 transactivation in Huh7 cells. However, dietary supplementation of female B6C3F1 mice with curcumin (500 mg/kg diet) for 2 weeks did not increase the hepatic PON1 mRNA and protein levels. No curcumin was detectable in the plasma of the 12 h fasted mice. In conclusion, curcumin may be a potent PON1 inducer in cultured cells in vitro, but not in the liver of curcumin-fed mice because of its low concentrations in vivo.

scholarly journals miR-338-5p Regulates the Viability, Proliferation, Apoptosis and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting NFAT5

2018 ◽  
Vol 49 (3) ◽  
pp. 899-910 ◽  
Author(s):  
Ting Guo ◽  
Hao Ding ◽  
Hui Jiang ◽  
Nirong Bao ◽  
Liwu Zhou ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Annexin V ◽  
Luciferase Reporter ◽  
G1 Arrest ◽  
Luciferase Reporter Gene ◽  
Activated T Cells ◽  
Aberrant Expression ◽  
Downstream Target ◽  
Gene Assay ◽  
Fibroblast Like Synoviocytes

Background/Aims: MicroRNAs (miRNAs) have been reported to be involved in Rheumatoid arthritis (RA) pathogenesis and prognosis. However, little is known about the disease mechanism in RA. Here, we aim to investigate the potential association between miR-338-5p and NFAT5 in RA. Methods: Aberrant expression of miR-338-5p in RA tissues and rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) compared to the normal were determined by RT-qPCR. Cell viability was determined using the CCK-8 assay, and cell apoptosis was analyzed via Annexin V-FITC/PI double staining and was detected using flow cytometry. The targeted relationship was determined by TargetScan database and dual luciferase reporter gene assay. Results: Upregulation of miR-338-5p facilitated the proliferation, migration, invasion and induced G0/G1 arrest of RAFLSs while miR-338-5p inhibitor functioned oppositely. Nuclear factor of activated T-cells 5 (NFAT5) was confirmed as a downstream target of miR-338-5p which expression was directly suppressed by miR-338-5p. Overexpression of NFAT5 attenuated the proliferation and metastasis of RAFLSs and those changes could be rescued by co-transfection of miR-338-5p. Conclusion: miR-338-5p promotes RAFLS’s viability and proliferation, migration by targeting NFAT5, suggesting a novel therapeutic strategy for RA.

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