• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.191-201
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• 2019

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or $GTP{\gamma}S$. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

• Journal of Korean Medicine Rehabilitation
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• v.32 no.2
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• pp.19-36
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• 2022

Objectives This study is to investigate the effects and mechanisms of Imyo-san (IMS) on the obese mice model induced by high-fat diet. Methods Antioxidative capacity was measured by in vitro method. C57BL/6 mice were randomly assigned into 5 groups (n=7). Normal group was fed general diet (Normal). The other 4 groups were fed high fat diet (HFD) with water (Control), with Garcinia gummi-gutta (GG, Garcinia gummi-gutta 200 mg/kg), with low-dose IMS (IMSL, Imyo-san 0.54 g/kg) and with high-dose IMS (IMSH, Imyo-san 1.08 g/kg). Results IMS showed high radical scavenging activity. After 6 week experiment, body weight, food intake, food efficiency ratio (FER), epididymal fat and liver weight, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol, sterol regulatory element-binding protein-1 (SREBP-1), phospho-acetyl-CoA carboxylase (p-ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), SREBP-2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), phospho-liver kinase B1 (p-LKB1), phospho-AMP-activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor 𝛼 (PPAR𝛼), peroxisome proliferator-activated receptor 𝛾 coactivator-1𝛼 (PGC-1𝛼), uncoupling protein-2 (UCP-2), carnitine palmitoyltransferase 1A (CPT-1A), and histology of liver and epididymal fat were measured and analysed. Body weight gain, FER, liver and epididymal fat weight of IMS groups were significantly decreased. There were significant improvements in blood lipids with less TG, TC, LDL-cholesterol, VLDL-cholesterol and more HDL-cholesterol. Proteins associated with lipid synthesis (SREBP-1, p-ACC, FAS, SCD-1) and cholesterol (SREBP-2, HMGCR) was improved. Factors regulating lipid synthesis and lipid catabolism (p-LKBI, p-AMPK, PPARα, PGC-1α, UCP-2, CPT-1A) were increased. In histological examinations, IMS group had smaller fat droplets than control group. All results increased depending on concentration. Conclusions It can be suggested that IMS has anti-obesity effects with improving lipid metabolism.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.3
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• pp.188-195
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• 2016

Neutrophils and lymphocytes are essential inflammatory cells in the pathogenesis of allergy. In this study, we evaluated the role of house dust mite (HDM) in the interaction between allergic lymphocytes and neutrophils. The extract of Dermatophagoides pteronissinus (DP) showed a stronger anti-apoptotic impact on neutrophil apoptosis in the coculture of allergic neutrophils with allergic lymphocytes when compared with that in allergic neutrophils alone. DP increased IL-6, IL-8, MCP-1, and GM-CSF in allergic lymphocytes, and the increased cytokines were inhibited by rottlerin-an inhibitor of the protein kinase C (PKC) ${\delta}$, as well as by SB202190-a p38 MAPK inhibitor. DP activated p38 MAPK in a time-dependent manner. The activation of p38 MAPK was suppressed by PAR2i, which is a protease-activated receptor (PAR) 2 inhibitor, and rottlerin. Both aprotinin-a serine protease inhibitor-and E64-a cysteine protease inhibitor-were not effective on cytokine secretion of lymphocytes. These results, despite increased cytokines in allergic lymphocytes via DP, did not show any differences between asthma and allergic rhinitis. Molecules, including cytokines, released by DP in lymphocytes inhibited the migration of neutrophils. This finding may further elucidate the pathogenic mechanism of allergic diseases due to HDM.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.1-14
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• 2009

Ovarian cancer is a significant cause of cancer-related death in women, but the main biological causes remain open questions. Hormonal factors have been considered to be an important determinant causing ovarian cancer. Recent studies have shown that gonadotropin-releasing hormone (GnRH)-I and its analogs have clinically therapeutic value in the treatment of ovarian cancer. In addition, numerous studies have shown that the potential of GnRH-II in normal reproductive system or reproductive disorder. GnRH-I receptors have been detected in approximately 80% of ovarian cancer biopsy specimens as well as normal ovarian epithelial cells and immortalized ovarian surface epithelium cells. GnRH-II receptors have also been found to be more widely expressed than GnRH-I receptors in mammals, suggesting that GnRH receptors may have additional functions in reproductive system including ovarian cancer. The signal transduction pathway following the binding of GnRH to GnRH receptor has been extensively studied. The activation of protein kinase A/C (PKA/PKC) pathway is involved in the GnRH-I induced anti-proliferative effect in ovarian cancer cells. In addition, GnRH-I induced mitogen-activated protein kinase (MAPK) activation plays a role in anti-proliferative effect and apoptosis in ovarian cancer cells and the activation of transcriptional factors related to cellular responses. However, the role of GnRH-I and II receptors, there are discrepancies between previous reports. In this review, the role of GnRH in ovarian cancer and the mechanisms to induce anti-proliferation were evaluated.

• Journal of Photoscience
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• v.9 no.2
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• pp.335-337
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• 2002

Blue light (BL) induces stomatal opening through activation of H$^{+}$ pump, which creates electrical gradient across the plasma membrane for $K^{+}$ uptake into guard cells. The pump is the plasma membrane H$^{+}$ -ATPase and is activated via phosphorylation of the C-terminus with concomitant binding of the 14-3-3 protein. The opening is initiated by the perception of BL through phototropin (phot), which are recently identified as BL receptors in stomatal guard cells. In this study, we provide the biochemical evidence for phots as BL receptors in stomatal guard cells. vfphot was phosphorylated reversibly by BL, and phosphorylation levels of vfphot increased earlier than those of the plasma membrane W-ATPase. BL-dependent phosphorylations of vfphot and H$^{+}$-ATPase showed similar fluence dependency. Staurosporin, an inhibitor of serine/threonine protein kinase, and diphenyleneiodonium chloride (DPI), an inhibitor of flavoprotein, inhibited BL-dependent phosphorylations of vfphot and H$^{+}$ -ATPase. These results indicate that vfphot acts as a BL-receptor mediating stomatal opening.l opening.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1170-1178
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• 2008

The cyclopentenone prostaglandins (cyPGs) prostaglandin $A_1$ ($PGA_1$) and 15-deoxy-${\Delta}^{12,14}$-prostaglandin $J_2$ (15d-$PGJ_2$) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of $PGA_1$ in lipopolysaccharide (LPS)-induced expression of inter leu kin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-$PGJ_2$ inhibited expression of LPS-induced IL-10, whereas $PGA_1$ increased LPS-induced IL-10 expression. This synergistic effect of $PGA_1$ on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous $PGA_1$ and LPS treatment ($PGA_1$/LPS), and did not require new protein synthesis. The synergistic effect of $PGA_1$ was inhibited by GW9662, a specific peroxisome proliferator-activated receptor ${\gamma}(PPAR{\gamma})$ antagonist, and Bay-11-7082, a NF-${\kappa}B$ inhibitor. The extracellular signal-regulated kinases (ERK) inhibitor PD98059 increased the expression of $PGA_1$/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, $PGA_1$ inhibited LPS-induced ERK phosphorylation. The synergistic effect of $PGA_1$ on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and $PGA_1$ increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun $NH_2$-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of $PGA_1$ on LPS-induced IL-10 expression is NF-${\kappa}B$-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/JNK signaling pathways, and also associated with the $PPAR{\gamma}$ pathway. Our data may provide more insight into the diverse mechanisms of $PGA_1$ effects on the expression of cytokine genes.

• Toxicological Research
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• v.34 no.1
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• pp.13-21
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• 2018

Anthocyanins are naturally occurring water-soluble polyphenolic pigments in plants that have been shown to protect against cardiovascular diseases, and certain cancers, as well as other chronic human disorders. However, the anti-obesity effects of anthocyanins are not fully understood. In this study, we investigated the effects of anthocyanins isolated from the fruit of Vitis coignetiae Pulliat on the adipogenesis of 3T3-L1 preadipocytes. Our data indicated that anthocyanins attenuated the terminal differentiation of 3T3-L1 preadipocytes, as confirmed by a decrease in the number of lipid droplets, lipid content, and triglyceride production. During this process, anthocyanins effectively enhanced the activation of the AMP-activated protein kinase (AMPK); however, this phenomenon was inhibited by the co-treatment of compound C, an inhibitor of AMPK. Anthocyanins also inhibited the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-${\gamma}$, CCAAT/enhancer-binding protein a and b, and sterol regulatory element-binding protein-1c. In addition, anthocyanins were found to potently inhibit the expression of adipocyte-specific genes, including adipocyte fatty acid-binding protein, leptin, and fatty acid synthase. These results indicate that anthocyanins have potent anti-obesity effects due to the inhibition of adipocyte differentiation and adipogenesis, and thus may have applications as a potential source for an anti-obesity functional food agent.

• Nutrition Research and Practice
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• v.15 no.5
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• pp.568-578
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• 2021

BACKGROUND/OBJECTIVES: Psidium guajava L. (guava) leaves have been shown to exhibit hypoglycemic and antidiabetic effects in rodents. This study investigated the effects of guava leaf extract on adipogenesis, glucose uptake, and lipolysis of adipocytes to examine whether the antidiabetic properties are mediated through direct effects on adipocytes. MATERIALS/METHODS: 3T3-L1 cells were treated with 25, 50, 100 ㎍/mL of methanol extract from guava leaf extract (GLE) or 0.1% dimethyl sulfoxide as a control. Lipid accumulation was evaluated with Oil Red O Staining and AdipoRed assay. Immunoblotting was performed to measure the expression of adipogenic transcription factors, fatty acid synthase (FAS), and AMP-activated protein kinase (AMPK). Glucose uptake under basal or insulin-stimulated condition was measured using a glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose. Lipolysis from fully differentiated adipocytes was measured by free fatty acids release into the culture medium in the presence or absence of epinephrine. RESULTS: Oil Red O staining and AdipoRed assay have shown that GLE treatment reduced lipid accumulation during adipocyte differentiation. Mitotic clonal expansion, an early essential event for adipocyte differentiation, was inhibited by GLE treatment. GLE inhibited the expression of transcription factors involved in adipocyte differentiation, such as peroxisome proliferator-activated receptor 𝛄 (PPAR𝛄), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein-1c (SREBP-1c). FAS expression was also decreased while the phosphorylation of AMPK was increased by GLE treatment. In addition, GLE increased insulin-induced glucose uptake into adipocytes. In lipid-filled mature adipocytes, GLE enhanced epinephrine-induced lipolysis but reduced basal lipolysis dose-dependently. CONCLUSIONS: The results show that GLE inhibits adipogenesis and improves adipocyte function by reducing basal lipolysis and increasing insulin-stimulated glucose uptake in adipocytes, which can be partly associated with antidiabetic effects of guava leaves.

• BMB Reports
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• v.33 no.6
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• pp.525-530
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• 2000

The nerve growth factor (NGF) induces neuronal differentiation and neurite outgrowth of PC12 cells, whereas epidermal growth factors (EGF) stimulate growth and proliferation of the cells. In spite of this difference, NGF-or EGF-treated PC12 cells share various properties in cellular-signaling pathways. These include the activation of the phosphoinositide (PI)-3 kinase, 70 kDa S6 kinase, and in the mitogen-activated protein (MAP) kinase pathway, following the binding of these growth factors to intrinsic receptor tyrosine kinases (RTKs). Therefore, many studies have been attempted to access the critical signaling events in determining the differentiation and proliferation of PC12 cells. In this study, we investigated the cytosolic phospholipase $A_2$ ($cPLA_2$) in neurite behavior in order to identify the differences of signaling pathways between the NGF-induced differentiation and the EGF-induced proliferation of PC12 cells. We have showed here that the $cPLA_2$ was translocated from cytosol to membrane only in NGF-treated cells. We also demonstrated that this translocation is associated with NGF-induced activation of phospholipase $C-{\gamma}(PLC-{\gamma})$, which elevates intracellular $Ca^{2+}$ concentration. These results reveal that the translocation of $cPLA_2$ may be a requisite event in the neuronal differentiation of PC12 cells. Various phospholipase inhibitors were used to confirm the importance of these enzymes in the differentiation of PC12 cells. Neomycin B, a PLC inhibitor, dramatically inhibited the neurite outgrowth, and two distinct $PLA_2$ inhibitors, 4-bromophenacyl bromide (BPB) and arachidonyltrifluoro-methyl ketone ($AACOCF_3$) also suppressed the neurite outgrowth of the cells, as well Taken together, these data indicated that $cPLA_2$ is involved in NGF-induced neuronal differentiation and neurite outgrowth of PC12 cells.

• Molecules and Cells
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• v.38 no.1
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• pp.58-64
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• 2015

Activating transcription factor-3 (ATF3) acts as a negative regulator of cytokine production during Gram-negative bacterial infection. A recent study reported that ATF3 provides protection from Streptococcus pneumoniae infection by activating cytokines. However, the mechanism by which S. pneumoniae induces ATF3 after infection is still unknown. In this study, we show that ATF3 was upregulated via Toll-like receptor (TLR) pathways in response to S. pneumoniae infection in vitro. Induction was mediated by TLR4 and TLR2, which are in the TLR family. The expression of ATF3 was induced by pneumolysin (PLY), a potent pneumococcal virulence factor, via the TLR4 pathway. Furthermore, ATF3 induction is mediated by p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Thus, this study reveals a potential role of PLY in modulating ATF3 expression, which is required for the regulation of immune responses against pneumococcal infection in macrophages.