• Journal of Ginseng Research
• /
• v.44 no.5
• /
• pp.690-696
• /
• 2020

Background: As the main metabolites of ginsenosides, 20(S, R)-protopanaxadiol [PPD(S, R)] and 20(S, R)-protopanaxatriol [PPT(S, R)] are the structural basis response to a series of pharmacological effects of their parent components. Although the estrogenicity of several ginsenosides has been confirmed, however, the underlying mechanisms of their estrogenic effects are still largely unclear. In this work, PPD(S, R) and PPT(S, R) were assessed for their ability to bind and activate human estrogen receptor α (hERα) by a combination of in vitro and in silico analysis. Methods: The recombinant hERα ligand-binding domain (hERα-LBD) was expressed in E. coli strain. The direct binding interactions of ginsenosides with hERα-LBD and their ERα agonistic potency were investigated by fluorescence polarization and reporter gene assays, respectively. Then, molecular dynamics simulations were carried out to simulate the binding modes between ginsenosides and hERα-LBD to reveal the structural basis for their agonist activities toward receptor. Results: Fluorescence polarization assay revealed that PPD(S, R) and PPT(S, R) could bind to hERα-LBD with moderate affinities. In the dual luciferase reporter assay using transiently transfected MCF-7 cells, PPD(S, R) and PPT(S, R) acted as agonists of hERα. Molecular docking results showed that these ginsenosides adopted an agonist conformation in the flexible hydrophobic ligand-binding pocket. The stereostructure of C-20 hydroxyl group and the presence of C-6 hydroxyl group exerted significant influence on the hydrogen bond network and steric hindrance, respectively. Conclusion: This work may provide insight into the chemical and pharmacological screening of novel therapeutic agents from ginsenosides.

• The Korean Journal of Nuclear Medicine
• /
• v.17 no.2
• /
• pp.35-40
• /
• 1983

To evaluated the usefulness of cultured human fibroblast for insulin receptor assay, the authors cultured fibroblast from biopsied normal adult female eyelid skin and assayed the insulin receptor with radioreceptor assay method. From the data obtained, percent of labeled insulin bound, numbers of insulin binding sites, affinity constants(Ka) and affinity of the empty sites(Ke) were calculated. The results were as follow; 1) The percent radioactivity bound of cultured fibroblast reached plateau at 4 hours $15^{\circ}C$ incubation. 2) The scatchard plot of insulin binding to cultured human fibroblast was curvilinear and the affinity to receptor was decreased with increased receptor occupancy. 3) The numbers of high affinity, low affinity and total insulin receptor of cultured fibroblasts were 852, 24,800 and 25,652 sites per cell. 4) High and low affinity constants of cultured fibroblasts were $3.4\times^{10}M^{-1},\;and\;1.08\times10^8M^{-1}$, and the affinity of empty site was $5.0\times10^8M^{-1}$.

• Proceedings of the Korean Biophysical Society Conference
• /
• 2003.06a
• /
• pp.66-66
• /
• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• Journal of Ginseng Research
• /
• v.43 no.3
• /
• pp.442-451
• /
• 2019

Background: Ginseng has a wide range of beneficial effects on health, such as the mitigation of minor and major inflammatory diseases, cancer, and cardiovascular diseases. There are abundant data regarding the health-enhancing properties of whole ginseng extracts and single ginsenosides; however, no study to date has determined the receptors that mediate the effects of ginseng extracts. In this study, for the first time, we explored whether the antiinflammatory effects of Rg3-enriched red ginseng extract (Rg3-RGE) are mediated by retinoid X receptor ${\alpha}$-peroxisome-proliferating receptor ${\gamma}$ ($RXR{\alpha}-PPAR{\gamma}$) heterodimer nuclear receptors. Methods: Nitric oxide assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, quantitative reverse transcription polymerase chain reaction, nuclear hormone receptor-binding assay, and molecular docking analyses were used for this study. Results: Rg3-RGE exerted antiinflammatory effects via nuclear receptor heterodimers between $RXR{\alpha}$ and $PPAR{\gamma}$ agonists and antagonists. Conclusion: These findings indicate that Rg3-RGE can be considered a potent antiinflammatory agent, and these effects are likely mediated by the nuclear receptor $RXR{\alpha}-PPAR{\gamma}$ heterodimer.

• YAKHAK HOEJI
• /
• v.38 no.5
• /
• pp.602-607
• /
• 1994

Based upon the previous experiments showing that kidney and lung tissues of rat had relatively abundant bradykinin binding sites, we tried to characterize and determine the densities of the bradykinin binding sites in the rabbit kidney tissue and proximal tubular cells under different growing conditions. Among the kidney tissue renal medulla segments showed the highest bradykinin binding sites. To determine which growth factors are to add in the serum free culture medium to express selectively the bradykinin binding sites in the rabbit kidney proximal tubular cells, we tried so called hormone-deletion approach and in here insulin, hydrocortisone, transferrin, triiodothyronine and prostaglandin $E_1$ are examined. By performing receptor binding assay and determination of protein concentrations, we may conclude that the most required hormones in the expression for bradykinin binding sites are insulin and transferrin, and fetal bovine serum is shown to be less effective in this regard.

• Natural Product Sciences
• /
• v.25 no.1
• /
• pp.28-33
• /
• 2019

A popular approach for the study of estrogen receptor ${\alpha}$ inhibition is to investigate the protein-protein interaction between the estrogen receptor (ER) and the coactivator surface. In our study, we investigated phytochemicals from Rubus coreanus that were able to disrupt $ER{\alpha}$ and coactivator interaction with an $ER{\alpha}$ antagonist. The E-screen assay and molecular docking analysis were performed to evaluate the effects of the estrogenic activity of R. coreanus extract and its constituents on the MCF-7 human breast cancer cell line. At $100{\mu}g/mL$, R. coreanus extract significantly stimulated cell proliferation ($574.57{\pm}8.56%$). Sanguiin H6, which was isolated from R. coreanus, demonstrated the strongest affinity for the $ER{\alpha}$ coactivator-binding site in molecular docking analysis, with a binding energy of -250.149. The initial results of the study indicated that sanguiin H6 contributed to the estrogenic activity of R. coreanus through the activation of the $ER{\alpha}$ coactivator-binding site.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.5
• /
• pp.375-377
• /
• 2000

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the hydrolysis of phosphatidyl-4,5-bisphosphate catalysed by phosphatidylinositol - specific phospholipase C yields to D-myo-inositol - 1,4,5-trisphosphate and diacylglycerol, which are well known second messengers. The binding of InsP$_3$to a receptor located on the endoplasmic reticulum triggers a calcium release from the endoplasmic reticulum. We have detected and partially characterised key components of phosphoinositide signaling. First, tobacco microsomal fraction and plasma membrane PI-PLC. Consecutively, using a radioligand binding assay we have identified a $Ca^{2+}$ -dependent high affinity InsP$_3$binding site in microsomal membrane fraction vesicle preparation and then we have measured inositol-1,4,5-trisphosphate induced calcium release from tobacco microsomal fraction. These findings suggest that phosphoinositide signaling system is present and operates in the tobacco suspension culture.e.

• Proceedings of the PSK Conference
• /
• 2001.10a
• /
• pp.218.1-218.1
• /
• 2001

• Korean Journal of Animal Reproduction
• /
• v.18 no.2
• /
• pp.95-99
• /
• 1994

The purpose of the present study was to determine the in vivo effect of oxytocin antagonist-I(Al) on uterine oxytocin receptor number (Rn) and/or binding affinity (Kd) in the estrous rat. Anesthetized rats were given a bolus infusion of control or 5${\mu}\textrm{g}$ of AI and sacri-ficed 0.5 and 4 hours later. The uterine tissue was removed, trimmed and frozen. Membrane oxytocin receptors were isolated after homogenization of uterine tissue and differential ultracentrifugation. The oxytocin receptor assay was performed by saturation with cold oxytocin competion with a high specific activity oxytocin antagonist. Rn and Kds were determined by nonlinear curve fitting methods. No differences(p>0.05) between the AI and control treated animals in either oxytocin receptor number or binding affinity was detected in this study. These data suggest that the major mode of action of AI is via competitive inhibition at the uterine oxytocin receptor and not by altering receptor number or binding affinity.

• BMB Reports
• /
• v.36 no.2
• /
• pp.207-213
• /
• 2003

Peroxisome proliferator-activated receptors (PPARs) are orphan nuclear hormone receptors that are known to control the expression of genes that are involved in lipid homeostasis and energy balance. PPARs activate gene transcription in response to a variety of compounds, including hypolipidemic drugs. Most of these compounds have high affinity to the ligand-binding domain (LBD) of PPARs and cause a conformational change within PPARs. As a result, the receptor is converted to an activated mode that promotes the recruitment fo co-activators such as the steroid receptor co-activator-1 (SRC-1). Based on the activation mechanism of PPARs (the ligand binding to $PPAR{\gamma}$ induces interactions of the receptor with transcriptional co-activators), we performed Western blot and ELISA. These showed that the indomethacin, a $PPAR{\gamma}$ ligand, increased the binding between $PPAR{\gamma}$ and SRC-1 in a ligand dose-dependent manner. These results suggested that the in vitro conformational change of $PPAR{\gamma}$ by ligands was also induced, and increased the levels of the ligand-dependent interaction with SRC-1. Collectively, we developed a novel and useful ELISA system for the mass screening of $PPAR{\gamma}$ ligands. This screening system (based on the interaction between $PPAR{\gamma}$ and SRC-1) may be a promising system in the development of drugs for metabolic disorders.