• Biomolecules & Therapeutics
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• v.13 no.2
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• pp.78-83
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• 2005

2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) has previously shown to induce neurotoxicity through intracellular $Ca^{2+}$ increase in rat neurons. In this study we investigated the role and signaling pathway of intracellular $Ca^{2+}$ in TCDD-induced inhibition of neuronal cell proliferation in SK-N-SH human neuronal cells. We found that TCDD(10nM) rapidly increased the level of intracellular $Ca^{2+}$, which was completely blocked by the extracellular $Ca^{2+}$ chelation with EGTA (1 mM) or by pretreatment of the cells with the non-selective cation channel blocker. flufenamic acid (200 ${\mu}M$). However, pretreatment of the cells with dantrolene (25 ${\mu}M$) and TMB-8(10 ${\mu}M$), intracellular $Ca^{2+}$-release blockers, or a voltage-sensitive $Ca^{2+}$ channel blocker, varapamil (100 ${\mu}M$), failed to block the TCDD-induced $Ca^{2+}$ increase in the cells. In addition, TCDD induced a rapid and transient activation of phatidvlinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2(ERK1/2), which was ingnificantly blocked by the pretreatment with BAPTA, an intracellular $Ca^{2+}$ chelator, and LY294002, a PI3K inhibitor. Furthermore, inhibitors of PI3K, ERK, or an intracellular $Ca^{2+}$ chelator further potentiated the anti-proliferative effect of TCDD in the cells. Collectively, the results suggest that intracellular $Ca^{2+}$ and PI3K-dependent activation of ERK 1/2 may be involved in the TCDD-induced inhibition of cell proliferation in SK-N-SH human neuronal cells.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• Journal of Ginseng Research
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• v.22 no.2
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• pp.126-132
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• 1998

In the present study, we examined effects of ginseng saponins (ginsenosides) on pp60c-src protein tyrosine kinase (PTK) activity, intracellular calcium concentration ([$Ca^{2+}$]i), and cell proliferation in NIH3T3 cells. Eight different ginsenosides [ginsenoside-Rb1 (G-$Rb_1$), -$Rb_2$, -Rc, -Rd, -Re, -Rf, -$Rg_1$, -$Rg_2$) and ginseng total saponin (GTS) were used for these experiments. All ginsenosides and GTS tested stimulated the activation of $pp60^{c-src}$ kinase, and especially G-$Rb_1$,-Rd,-$Rg_1$, and -$Rg_1$ showed a higher stimulatory effect than others at 16.7 $\mu\textrm{g}$/ml of ginsenosides with a 18 hr-incubation, increasing the activity by 4.5, 3.5, 3.5, and 3.0-fold, respectively, over that of untreated control. In addition, both G-Rd and -$Rg_2$)Rg2 increased ($Ca^{2+}$), to 202 and 334 nM, respectively, about 2-3-fold above the basal level within 7min at 250 $\mu\textrm{g}$/yml of ginsenosides. The increases of ($Ca^{2+}$), were eliminated by Pretreatment of EGTA, an extracellular calcium chelator, suggtasting that they result from an influx of calcium ion from extracellular medium rather than an efflux from intracellular calcium store, endoplasmic reticulum (ER). All ginsenosides studied enhanced cell proliferation to 1.2-1.4-fold over that of untreated control at 5~250 $\mu\textrm{g}$/ml of concentrations. Interestingly the promotion of cell proliferation by ginsenosides corresponded with the activation of c-src kinase, which is an early step in the mitogenic signaling cascade. Taken together, we suggest that some ginsenosides may lead to cellProliferation via the activation of cellular signal transduction Pathway involving $pp60^{c-src}$ kinase.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.5
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• pp.449-457
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• 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though $Ca^{2+}$ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ($[Ca^{2+}]_i$) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on $[Ca^{2+}]_i$ in human neutrophils. We observed that NAC ($1{\mu}M{\sim}1mM$) and cysteine ($10{\mu}M{\sim}1mM$) increased $[Ca^{2+}]_i$ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in $[Ca^{2+}]_i$ in human neutrophils was observed. In $Ca^{2+}$-free buffer, NAC- and cysteine-induced $[Ca^{2+}]_i$ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in $[Ca^{2+}]_i$ in human neutrophils occur through $Ca^{2+}$ influx. NAC- and cysteine-induced $[Ca^{2+}]_i$ increase was effectively inhibited by calcium channel inhibitors SKF96365 ($10{\mu}m$) and ruthenium red ($20{\mu}m$). In $Na^+$-free HEPES, both NAC and cysteine induced a marked increase in $[Ca^{2+}]_i$ in human neutrophils, arguing against the possibility that $Na^+$-dependent intracellular uptake of NAC and cysteine is necessary for their $[Ca^{2+}]_i$ increasing activity. Our results show that NAC and cysteine induce $[Ca^{2+}]_i$ increase through $Ca^{2+}$ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• The Journal of Internal Korean Medicine
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• v.31 no.4
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• pp.693-705
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• 2010

Objectives : The water extract of Daechilgi-tang(DCGT) has traditionally been used for treatment of qi stagnation(氣滯), which is considered to be one of the important causes of neuronal disease in oriental medicine. However, little is known about the mechanism by which DCGT protects neuronal cells from brain cell damages. Methods and Results : The author tested the mechanism of the cytoprotective effect of DCGT on glutamate -stimulated rat C6 glial cells. DCGT significantly protected C6 glial cells from glutamate in MTT assay. Pre-treatment of C6 glial cells with DCGT markedly inhibited the DNA fragmentation of C6 cells induced by glutamate. Glutamate increased the generation of reactive oxygen species(ROS) and intracellular calcium level in C6 glial cells. However, pre-treatment with DCGT markedly suppressed the increase of ROS generation and intracellular calcium accumulation induced by glutamate. Among apoptosis signaling mediators, DCGT markedly increased the expression level of Bcl2 in glutamate-treated cells. It also inhibited the cleavage of caspase-3 and PARP proteins by glutamate in C6 glial cells. Conclusions : These results suggest that DCGT protects brain cells from glutamate cytotoxicity through inhibition of ROS generation and activation of apoptosis signaling pathway as well as induction of the anti-oxidant system.

• Biomolecules & Therapeutics
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• v.23 no.6
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• pp.539-548
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• 2015

Prostaglandin $E_2$ ($PGE_2$), a major product of cyclooxygenase, binds to four different prostaglandin $E_2$ receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of $PGE_2$ induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and ${\beta}$-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway.

• The Korean Journal of Pain
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• v.31 no.2
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• pp.73-79
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• 2018

All drugs have both favorable therapeutic and untoward adverse effects. Conventional opioid analgesics possess both analgesia and adverse reactions, such as nausea, vomiting, and respiratory depression. The opioid ligand binds to ${\mu}$ opioid receptor and non-selectively activates two intracellular signaling pathways: the G protein pathway induce analgesia, while the ${\beta}$-arrestin pathway is responsible for the opioid-related adverse reactions. An ideal opioid should activate the G protein pathway while deactivating the ${\beta}$-arrestin pathway. Oliceridine (TRV130) has a novel characteristic mechanism on the action of the ${\mu}$ receptor G protein pathway selective (${\mu}$-GPS) modulation. Even though adverse reactions (ADRs) are significantly attenuated, while the analgesic effect is augmented, the some residual ADRs persist. Consequently, a G protein biased ${\mu}$ opioid ligand, oliceridine, improves the therapeutic index owing to increased analgesia with decreased adverse events. This review article provides a brief history, mechanism of action, pharmacokinetics, pharmacodynamics, and ADRs of oliceridine.

• The Journal of Korean Medicine
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• v.32 no.1
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• pp.109-120
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• 2011

Objectives: The aim of this investigation was to evaluate the efficacy of KHchunggan-tang aqueous extract on the experimental nonalcoholic fatty liver disease(NAFLD) induced by palmitate. Materials and Methods: To generate a cellular model of NAFLD, we used HepG2 cells, a human hepatoma cell line, treated with 0.5 mM palmitate. By this cellular model, effects of KHchunggan-tang aqueous extract were evaluated. Intracellular lipid accumulation, free radical formation, and apoptosis were detected by Nile red staining, 2',7'-dichloroflourescin diacetate(H2DCF-DA), and 4',6-diamidino-2-phenylindole(DAPI)/propidium iodide(PI) staining, respectively. Some proteins related with NAFLD were determined by western blot. Results: Typical pathological features of NAFLD occurred in the cellular model. Palmitate increased the levels of intracellular lipid vacuoles, decreased cell viability, and increased apoptosis. Palmitate increased free radical formation and lipid peroxidation, too. However, KHchunggan-tang aqueous extract reduced palmitate-induced pathologic features, i.e. steatosis, free radical formation, and apoptosis. In addition, KHchunggan-tang aqueous extract suppressed palmitate-activated c-Jun N-terminal kinase(JNK) signaling, and SP600125, a JNK inhibitor, significantly reversed the palmitate-induced pathologic changes as KHchunggan-tang aqueous extract. It means that the signaling pathway other than JNK can be involved in the KHchunggan-tang mediated cellular protection of palmitate-treated Hep G2 cells. Conclusions: These results suggest that KHchunggan-tang aqueous extract has hepatoprotective effects on NAFLD with combined properties in cellular steatosis, ROS production, and cytoprotection, and thus may have valuable clinical applications for treatment of this chronic liver disease.

• Journal of Applied Biological Chemistry
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• v.62 no.4
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• pp.347-353
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• 2019

Stings of jellyfish, which frequently occur in a warm season, cause severe pain, inflammation and sometimes irreversible results such as the death. Harmful venoms from jellyfish, therefore, have been studied for finding the therapeutic agents to relieve pain or to neutralize toxic components. However, it is still unclear if and how jellyfish venom reveal neuronal toxicity even though pain induction seems to result from the activation of nociceptors such as nerve endings. In this study, using HT-22 cell line, we investigated neurotoxic effects of the venom of Chrysaora pacifica (CpV) which appears in South-East ocean of Korea. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, CpV significantly reduced the viability of HT-22 cells in a dose-dependent manner. Additionally, in 2',7'-Dichlorofluorescin diacetate fluorescence test under the culture condition lacking dominant inflammatory factors, CpV remarkably increased the production of intracellular reactive oxygen species (ROS). Reduced responsive fluorescence to Rhodamine123 and increased expression of intracellular cytochrome c were also observed in HT-22 cells treated with CpV. These indicate that CpV-reduced viability of HT-22 cells may be due to the activation of apoptotic signalings mediated with oxidative stress and mitochondrial dysfunction. Furthermore, removing Ca²⁺ ion or adding N-acetyl-Lcystein remarkably blocked the CpV effect to reduce the viability of HT-22 cells. The findings in this study clearly demonstrate that CpV may activate Ca²⁺-mediated ROS signalings and mitochondrial dysfunction resulting in neuronal damage or death, and suggest that blocking Ca²⁺ pathway is a therapeutic approach to possibly block toxic effects of jellyfish venoms.