• Journal of Nutrition and Health
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• v.53 no.5
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• pp.452-463
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• 2020

Purpose: Aster tataricus (AT) is one of the Asteraceae perennial herbs used in traditional Chinese medicine. The herb contains various bioactive substances, such as flavonoids, isoflavonoids, and phenolic compounds in the roots, and exhibits a range of effects including anti-bacterial, anti-oxidant, and anti-inflammatory activities. This study compared the immunomodulatory effects of ethanol and water extracts of whole AT, except the roots, and analyzed the molecular mechanisms for the regulatory effects on cytokine secretion from THP-1 cells. Methods: The effects of AT extract on the cell viability and proliferation of THP-1 cells were analyzed using the Cell Counting Kit-8 method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of the AT-treated THP-1 cells were measured using an enzyme-linked immunosorbent assay. The protein levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor kappa B (IκBα), and mitogen-activated protein kinase (MAPK) phosphorylation in the cell lysates were determined by western blotting. Results: The water extract and the ethanol extract of AT did not affect the cell viability, and increased the proliferation of THP-1 cells significantly compared to the vehicle. The water extract increased the secretion of IL-1β from THP-1 cells in a dose-dependent manner, but the ethanol extract had no effect. The expression of COX-2 and iNOS protein and the phosphorylation of MAPK and Akt were induced in AT-treated cells. In addition, IκBα was degraded by AT in a concentration-dependent manner. IL-1β secretion by AT was reduced by extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors, while TNF-α secretion was decreased by inhibitors of ERK, p38 MAPK, and JNK. Interestingly, the p38 MAPK inhibitor increased the production of IL-1β by AT further. Conclusion: The water extract of the above-ground parts of AT contains immunomodulatory bioactive substances that stimulate immune cells through the MAPK signaling pathway.

• Journal of Life Science
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• v.28 no.4
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• pp.435-443
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• 2018

AMP-activated protein kinase (AMPK) functions as a metabolic master through regulating and restoring cellular energy balance. In skeletal muscle, AMPK increases myofibril protein degradation through the expression of muscle-specific ubiquitin ligases. Mori Folium, the leaf of Morus alba, is a traditional medicinal herb with various pharmacological functions; however, the effects associated with muscle atrophy have not been fully identified. In this study, we confirmed the effects of AMPK activation by examining the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, on the induction of atrophy and expression of atrophy-related genes in C2C12 myotubes. We also investigated the effects of the ethanol extract of Mori Folium (EEMF) on the recovery of AICAR-induced muscle atrophy in C2C12 myotubes. It was found that exposure to AICAR resulted in the stimulation of Forkhead box O3a (FOXO3a); an up-regulation of muscle-specific ubiquitin ligases such as Muscle Atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and a down-regulation of muscle-specific transcription factors, such as MyoD and myogenin; with the activation of AMPK. In addition, AICAR without cytotoxicity indicated a decrease in diameter of C2C12 myotubes. However, treatment with EEMF significantly suppressed AICAR-induced muscle atrophy of C2C12 myotubes in a dose-dependent manner as confirmed by a decrease in myotube diameter, which is associated with a reversed stimulation of FOXO3a by the inhibition of AMPK activation. These results indicate that the activation of AMPK by AICAR induces muscle atrophy, and EEMF has preeminent effects on the inhibition of AICAR-induced muscle atrophy through the AMPK signaling pathway.

• Journal of Life Science
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• v.25 no.12
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• pp.1384-1392
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• 2015

Sagantang (SGT), a Korean multiherb formula comprising six medicinal herbs, Paeonia lactiflora Pall., Belamcanda chinensis (L.) DC, Gardenia jasminoides Ellis, Poria cocos Wolf, Cimicifuga heracleifolia Komarov, and Artractylodes japonica Koidzumi, was recorded in “Dongeuibogam.” The present study investigated the anticancer potential of SGT in AGS human gastric carcinoma cells. The results indicated that SGT treatment significantly inhibited the growth and viability of AGS cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, in addition to chromatin condensation and DNA fragmentation, and the accumulation of annexin-V positive cells. The induction of apoptotic cell death by the SGT treatment was associated with up-regulation of Fas protein expression, truncation of Bid, and down-regulation of the anti-apoptotic Bcl-2 protein. The SGT treatment also effectively induced the loss of mitochondrial membrane potential, which was associated with the activation of caspases (caspase-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase. However, a pan-caspase inhibitor significantly blocked the SGT-induced apoptosis and growth suppression in AGS cells. This study suggests that SGT induces caspase-dependent apoptosis through an extrinsic pathway by upregulating Fas, as well as through an intrinsic pathway by modulating Bcl-2 family members in AGS cells. The results suggest that SGT may be a potential chemotherapeutic agent for the control of human gastric cancer cells. However, further studies will be needed to confirm the potential of SGT in cancer prevention and therapy in an in vivo model and to identify biological active compounds of SGT.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.5
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• pp.562-571
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• 2017

In this study, we investigated the anti-oxidant activities [electron-donating ability (EDA), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability, and reactive oxygen species (ROS) inhibitory activity], anti-wrinkle activities [collagenase inhibitory activity, suppression and/or phosphorylation of matrix metalloproteinases (MMPs), and mitogen-activated protein (MAP) kinase activity], and mRNA expression levels using reverse transcription-polymerase chain reaction (RT-PCR) assay in ultraviolet (UV) B ray ($50mJ/cm^2$)-irradiated human keratinocyte HaCaT cells. Josaengheugchal, Sinneungheugchal (SE), Shintoheug rice, Heugjinju rice, and Heugseol (HE) among colored rice varieties were reported to have excellent antioxidant properties. In the EDA and ABTS radical scavenging assays, extracts of the five colored rice varieties had scavenging activities of 72% at concentrations higher $50{\mu}g/mL$. In the collagenase inhibition assay, ethanol extracts of the five colored rice varieties showed high inhibitory effects of about 60% at concentrations higher $25{\mu}g/mL$. In the ROS inhibition assay, ethanol extracts of HE and SE showed very excellent inhibition efficacies at all concentrations. We determined molecular biological mechanisms of MMPs (MMP-1, -3, -8, and -13) and mitogen-activated protein kinase (MAPK) with HE, and the results show that HE suppressed expression of MMPs and phosphorylation of MAPK and increased expression of pro-collagen type I in UVB-irradiated cells. It was also confirmed by RT-PCR that HE reduced expression of MMPs mRNA. Therefore, these results suggest that HE has anti-wrinkle and collagen production effects and may be used as a material in the development of functional food and cosmetic industries.

• Journal of Life Science
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• v.31 no.12
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• pp.1110-1119
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• 2021

The purpose of this study was to investigate whether the inflammatory response in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages could be promoted by particulate matter 2.5 (PM_2.5) stimulation. To this end, the levels of inflammatory parameters, reactive oxygen species (ROS) and inflammation-regulating genes were investigated in RAW 264.7 cells treated with PM_2.5 in the presence or absence of LPS. Our results showed that the production levels of pro-inflammatory mediators (nitric oxide and prostaglandin E₂) and cytokines (interleukin-6 and -1β) were significantly increased by PM_2.5 stimulation in LPS-treated RAW 264.7 cells, which was correlated with increased expression genes involved in their production. In addition, when LPS-treated RAW 264.7 cells were exposed to PM_2.5, nuclear factor-kappaB (NF-κB) expression was further increased in the nucleus, and the expression of inhibitor of NF-κB as well as NF-κB in the cytoplasm was decreased. These results suggest that the co-treatment of PM_2.5 and LPS further increases the activation of the NF-κB signaling pathway compared to each treatment alone, thereby contributing to the promotion of transcriptional activity of inflammatory genes. Furthermore, although the generation of ROS was greatly increased by PM_2.5 in LPS-treated RAW 264.7 cells, the NF-κB inhibitor did not reduce the generation of ROS. In addition, when the generation of ROS was artificially suppressed, the production of inflammatory mediators and the activation of NF-κB were both abolished. Therefore, our results suggest that the increase in the NF-κB-mediated inflammatory response induced by PM_2.5 in LPS-treated RAW 264.7 macrophages was a ROS generation-dependent phenomenon.

• Journal of the Korean Chemical Society
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• v.67 no.2
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• pp.81-88
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• 2023

The contribution of hydrophobic residues to the protein folding reaction was studied by using HubWA variant proteins with I and L to V mutation. Folding kinetics of all V variant proteins was observed to be satisfied by a three-state on-pathway mechanism, U ⇌ I ⇌ N, where U, I, and N represent unfolded, intermediate, and native state, respectively. Three-state folding reaction was quantitatively analyzed and the free energy of folding of each elementary reactions and overall folding reaction, ΔG^o_UI, ΔG^o_IN, and ΔG^o_UN, were obtained. From the ratio of free energy difference between the variant protein and HubWA, ΔΔG^o_UI/ΔΔG^o_UN (ΔΔG^o_UI = ΔG^o_UI (variant protein) - ΔG^o_UI (HubWA) and ΔΔG^o_UN = ΔG^o_UN (variant protein) - ΔG^o_UN(HubWA)), the contribution of hydrophobic residues to HubWA folding was analyzed. The residues which are located in the hydrophobic core between α-helix and β-sheet, I3, I13, L15, I30, L43, I61 and L67, showed ΔΔG^o_UI/ΔΔG^o_UN value of ~0.5 when each of these residues was mutated to V, indicating that these residues form relatively solid hydrophobic core in the intermediate state. Residues located at the end of secondary structures and loop, I23, L69 and I36 showed ΔΔG^o_UI/ΔΔG^o_UN value below 0.4 when each of these residues was mutated to V, indicating that the region containing these residues are loosely formed in the intermediate state. V17A, L50V and L56V showed fairly high ΔΔG^o_UI/ΔΔG^o_UN value of ~0.8. Since L50 and L56 are located in the region containing long loop (residue 46 to 62), it is suggested that the high ΔΔG^o_UI/ΔΔG^o_UN value of these residues prevents the formation of aggregate at the early stage of folding reaction.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Journal of Physiology & Pathology in Korean Medicine
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• v.28 no.5
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• pp.530-536
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• 2014

Root barks of Paeonia suffruticosa Andrews (PS) was reported as contraindicated drugs of pregnancy by many Korean medical classics. Recently, a major ingredient component of PS, paeonol was reported that has contraceptive effect on early pregnancy in rats. However, the accurate molecular mechanism is not clear. In this study, we showed that PS decreased the expression of receptor for leukemia inhibitory factor (LIFR) in human endometrial Ishikawa cells at non-toxic dose, although the expression of leukemia inhibitory factor (LIF) was increased by PS. In addition, PS inhibited the adhesion of human trophoblastic JAR cells onto Ishikawa cells. Given importance of LIF-LIFR signaling pathway in the process of embryo implantation, the decreased LIFR expression by PS will be a good explanation on the PS- or its ingredient compounds-induced contraception.

• Molecules and Cells
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• v.26 no.5
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• pp.468-473
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• 2008

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that $Cl^-$ channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of $Cl^-$ channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. $Cl^-$ channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated $Cl^-$ concentration, as judged by flow cytometry analysis using MQAE as a $Cl^-$-detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS ($Cl^-$ channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive $Cl^-$-channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

• BMB Reports
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• v.40 no.5
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• pp.749-756
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• 2007

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5$\alpha$, Tssk5$\beta$, Tssk5$\gamma$ and Tssk5$\delta$. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5$\alpha$ which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5$\alpha$, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5$\alpha$ exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5$\beta$, TSSK5$\gamma$ and TSSK5$\delta$ cannot be stimulated at the CREB/CRE responsive pathway in comparison to TSSK5$\alpha$. These findings suggest that TSSK5$\beta$, TSSK5$\gamma$, TSSK5$\delta$ may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5$\beta$, TSSK5 $\gamma$ and TSSK5$\delta$ can directly interact with TSSK5$\alpha$. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.