• ALGAE
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• v.29 no.4
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• pp.355-366
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• 2014

Fucoxanthin is known to be an effective cell proliferation inhibitor with anti-tumor and anti-angiogenic activities. However, there is a lack of data regarding the biological effects of cis isomers of fucoxanthin. To assess the potential therapeutic properties of 9'-cis-(6'R) fucoxanthin (FcA), and 13-cis and 13'-cis-(6'R) fucoxanthin complex (FcB) isolated from Sarggassum siliquastrum, we investigated their inhibitory effects on matrix metalloproteinases (MMPs) in phorbol 12-myristate 13-acetate (PMA)-induced human fibrosarcoma (HT1080) cells. FcA and FcB reduced MMP-2 and MMP-9 protein and mRNA levels, as well as the migration of these cells, in a dose-dependent manner. Additionally, FcA and FcB increased levels of MMPs inhibition factors such as tissue inhibitor of metalloproteinase-1. FcA and FcB significantly inhibited the transcriptional activity of nuclear factor ${\kappa}B$ (NF-${\kappa}B$) and by inhibiting c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases. Our results demonstrate that suppression of the NF-${\kappa}B$, JNK, and p38 signaling pathways may inhibit PMA-induced MMP-2 and MMP-9 activity. Therefore, FcA and FcB may be useful in noninvasive therapeutic strategies against fibrosarcoma metastasis.

• Journal of Life Science
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• v.13 no.5
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• pp.551-558
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• 2003

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and PLD has been also reported to be overexpressed and hyperactivated in some human cancer. The aim of this study was to understand how PLD can be regulated in HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that in unstimulated state, basal PLD activity was higher in NIH3T3 cells overexpressing HCV core protein than in vector-transfected cells. Although expression of PLD and protein kinase C (PKC) in core protein-transformed cells was similar with that of control cells, phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated significantly PLD activity in core protein-transformed cells, compared with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor, and PKC translocation experiment showed that PKC-$\delta$ was mainly involved in the PMA-induced PLD activation in the core-transformed cells. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

• Biomedical Science Letters
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• v.18 no.2
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• pp.104-111
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• 2012

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which degrade extracellular matrix (ECM) during embryogenesis, wound healing, and tissue remodeling. Dysregulation of MMP activity is also associated with various pathological inflammatory conditions. In this study, we examined the expression pattern of MMPs during PMA-induced differentiation of THP-1 monocytic cells into macrophages. We found that MMP1, MMP8, MMP3, MMP10, MMP12, MMP19, MMP9, and MMP7 were upregulated during differentiation whereas MMP2 remained unchanged. Expression of MMPs increased in a time-dependent manner; MMP1, MMP8, MMP3, MMP10, and MMP12 increased beginning at 60 hr post PMA treatment whereas MMP19, MMP9, and MMP7 increased beginning at 24 hr post PMA treatment. To identify signal transduction pathways involved in PMA-induced upregulation of MMPs, we treated PMA-differentiated THP-1 cells with specific inhibitors for PKC, MEK1, NF-${\kappa}B$, PI3K, p38 MAPK and PLC. We found that inhibition of the MEK1 pathway blocked PMA-induced upregulation of all MMPs to varying degrees except for MMP-2. In addition, expression of select MMPs was inhibited by PI3K, p38 MAPK and PLC inhibitors. In conclusion, we show that of the MMPs examined, most MMPs were up-regulated during differentiation of monocyte into macrophage via the MEK1 pathway. These results provide basic information for studying MMPs expression during macrophage differentiation.

• Journal of Life Science
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• v.19 no.12
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• pp.1808-1814
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• 2009

In our previous study, a new herbal preparation (HemoHIM) was developed as a functional food for the radioprotection and immunomodulatory agents. In order elucidate the mechanism involved, we examined the effect of HemoHIM on the compound 48/80-induced histamine release, and on the phorbol 12-myristate 13-acetate (PMA)/calcium ionophore (A23187)-induced inflammatory cytokine secretion in HMC-1. The cell culture supernatants were harvested, and the cytokines (IL-4, IL-6, IL-8, TNF-$\alpha$, GM-CSF) in the supernatants were measured by enzyme-linked immunosorbent assay. The total RNA of the cells was extracted, and the cytokines or c-kit/tryptase/Fc$\varepsilon$RI's messenger RNA expressions were examined using reverse transcriptase polymerase chain reaction. Under low concentrations, HemoHIM inhibited histamine release in HMC-1 stimulated compound 48/80. Furthermore HemoHIM inhibited PMA/A23187-induced inflammatory cytokines' secreation or mRNA expression in a dose-dependent manner. But IL-8 secretion was not inhibited by low concentrayion of HemoHIM, respectively. The mRNA expression of c-kit and Fc$\varepsilon$RI were also inhibited in a dose-dependent manner. Tryptase mRNA expression was only inhibited by low concentration of HemoHIM. These results indicated that HemoHIM might be an useful agent for protection against allergy as well as immune modulation, especially since it is a relatively nontoxic natural product.

• The Korean Journal of Pharmacology
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• v.29 no.1
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• pp.121-130
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• 1993

This study was done to classify the proteins involved in the specific phosphorylation using the rat peripheral blood lymphocytes (rPBL) stimulated with mitogens, phorbol 12-myristate 13-acetate (PMA) and concanavalin A (Con A). The lymphocytes were incubated with $^{32}P-orthophosphate$ before PMA or Con A stimulation. The migration patterns of the phosphorylated proteins of mitogen-treated rPBL in two dimensional electrophoretic fields were analyzed after autoradiography. The stimulation of the lymphocytes with PMA and Con A increased the phosphorylation of thirteen protein fractions. The phosphorylation intensities of the protein spots differ to the treatments of the cells with specific kinase inhibitors, H-7 and W-7. These protein fractions were grouped into 3 classes, namely, PKC-mediated, CaM kinase-mediated, and other kinase mediated proteins. The effect of the duration of the stimulation on the phosphorylated behaviors occurred concurrently, not sequentially, although each individual protein fraction had a different time for the peak phosphorylation during the stimulation period upto 30 minutes. The phosphoproteins found in the cytosolic soluble fraction were phosphorylated prior to those in the pellet, whose phosphorylations were sustained at a high level for over 10 minutes. The above results suggest that the early events in lymphocyte activation involve 3 different sets of proteins which are phosphorylated by CaM kinase, PKC and other kinases, and those kinases do not work sequentially, but rather, independently or cooperatively.

• The Journal of Korean Obstetrics and Gynecology
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• v.34 no.3
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• pp.29-48
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• 2021

Objectives: Banchong-san (BCS) is a herbal formula composed of 13 korean medicinal herbs and is traditionally used to treat inflammatory diseases and pain. The object of this study was to research the antioxidant, anti-inflammatory, antipruritic and antimicrobial effects of the BCS in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods: In this experiment, effects of BCS on the following four were measured as follows: (1) Anti-oxidative effects were evaluated by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) Radical scavenging activity, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) Radical scavenging activity. (2) Anti-inflammatory effects were evaluated by the production amount of Reactive oxygen species (ROS), Nitric oxide (NO), Interleukin-1β (IL-1β), Interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), Prostaglandin E2 (PGE₂), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)(the previous two are "mRNA"), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (p38), inhibitor of nuclear factor kappa B (IκBα), nuclear factor kappa B (NF-κB) (the previous five are "Protein") in LPS-Stimulated RAW 264.7 cells. (3)Antipruritic effects were evaluated by the production amount of histamine, Leukotriene B4 (LTB4), LeukotrieneC4 (LTC4) Levels in phorbol 12-myristate 13-acetate(PMA)/ionomycin-stimulated MC/9 mast cell. (4) Anti-microbial effects were evaluated by the growth suppression of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus niger. Results: The following results were obtained through each measurement: (1) DPPH Radical Scavenging Activity, ABTS Radical Scavenging Activity evoked a significant concentration-dependent increase. (2) ROS, NO, IL-1β, IL-6, TNF-α, PGE₂ production amount, iNOS, COX-2 mRNA expression were significantly reduced in the BCS extraction group compared with the control group and significantly decreased the amount of ERK, JNK, p38, NF-κB Protein expression. The amount of IκB-α Protein Expression have increased significantly. (3) The amounts of histamine, LTB4, LTC4 were significantly decreased. (4) The antibacterial efficacy, BCS inhibited the growth of Escherichia coli, Pseudomonas aeruginosa at concentrations of 5 ㎍/ml, but did not suppress the growth of staphylococcus aureus and aspergillus niger. Conclusions: The experimental results show that BCS has anti-oxidant, anti-inflammatory, antipruritic and antimicrobial properties.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.187-190
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• 2004

Protein kinase C exists as a family of serine/threonine kinases which are broadly classified into three groups as cPKC nPKC and aPKC depending on their cofactor requirements. Previous studies have shown that the role of PKC in the process of mouse oocyte maturation. For example, phorbol 12-myristate 13-acetate which is known as an activator of cPKC and nPKC inhibits germinal vesicle break down and 1st polar body extrusion in maturing oocytes. In this study, the effect of thymeleatoxin, a specific activator of cPKC not nPKC, was tested comparing with PMA to address the roles of cPKC and nPKC during mouse oocyte maturation. Cumulus-oocyte complex were cultured in M16 medium for 6 or 12 hr with each of these PKC activators to investigate the effect of germinal vesicle breakdown (GVBD) or the extrusion of 1st polar body. IC/sup 50/ of GVBD were at concentrations of 50nM in PMA and 400nM in thymeleatoxin and of 1st polar body extrusion were 20nM in PMA and 200nM in thy- meleatoxin. The results suggest that activation of nPKC is more closely related to the inhibition of GVBD and 1st polar body extrusion than activation of cPKC. Additionally, we found that the oocytes inhibited 1st polar body extrusion with PMA or thymeleatoxin were arrested in metaphase I of first meiosis.

• Journal of Ginseng Research
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• v.39 no.2
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• pp.169-177
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• 2015

Background: Ginsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of $Ca^{2+}$ influx.We intended to explore the effects of GSRd on L-type $Ca^{2+}$ current ($I_{Ca,L}$) and define the mechanism of the suppression of $I_{Ca,L}$ by GSRd. Methods: Perforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results: (1) GSRd reduced $I_{Ca,L}$ peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration $(IC_{50})=32.4{\pm}7.1{\mu}mol/L$] and up-shifted the current-voltage (I-V) curve. (2) GSRd ($30{\mu}mol/L$) significantly changed the steady-state activation curve of $I_{Ca,L}$ ($V_{0.5}:-19.12{\pm}0.68$ vs. $-6.26{\pm}0.38mV$; n = 5, p < 0.05) and slowed down the recovery of $I_{Ca,L}$ from inactivation [the time content (${\zeta}$) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd ($100{\mu}mol/L$) was identified in perforated-patch recording when compared with whole-cell recording [$65.7{\pm}3.2%$ (n = 10) vs. $31.4{\pm}5.2%$ (n = 5), p < 0.01]. (4) Pertussis toxin ($G_i$ protein inhibitor) completely abolished the $I_{Ca,L}$ inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [$55{\pm}7.8%$ (n = 5) vs. $17.2{\pm}3.5%$ (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-$\small{L}$-cysteine (a nitric oxide scavenger) partly recovered the $I_{Ca,L}$ inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. Conclusion: These findings suggest that GSRd could inhibit $I_{Ca,L}$ through pertussis toxin-sensitive G protein ($G_i$) and a nitric oxide-cyclic guanosine monophosphate-dependent mechanism.

• Journal of Life Science
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• v.17 no.11
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• pp.1492-1496
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• 2007

In vitro IgE class switching could be induced through co-culture of CD40L-expressing KU812 cells and CD40-expressing B cells in the presence of IL-4 or IL-13. It has been generated several B cell lines, which produce rice allergen (RA)-specific IgM antibody by in witγo immunization (IVI) using peripheral blood lymphocyte (PBL). In this study, induction of RA-specific IgE antibody by KU812 cells was attempted. Before co-culture, we determined the CD40 expression in RA-specific B cell lines, RA9G11 and the CD40 ligand (CD40L) expression in activated KU812 cells by treatments with phorbol myristate acetate (PMA) and ionomycin for 6 hrs. Flow cytometric analysis shown that RA9G11 and activated KU812 cells expressed high level of CD40 and CD40L, respectively. RA9G11 cells were cultured with activated KU812 cells for 12 days in the presence of IL-4 for IgE class switching. Mature $C{\varepsilon}$ mRNA level and RA-specific IgE spot forming cells (SFC) were observed in all culture condition, and especially, high level of RA-specific IgE synthesis was determined the same ratio of RA9G11 and activated KU812 cells in the presence of 50U IL-4. Therefore, induction of RA-specific IgE synthesis by activated KU812 cells can be contributed in the application for allergic therapy and prevention.

• Molecules and Cells
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• v.38 no.12
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• pp.1064-1070
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• 2015

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10-100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO ($0.1-0.5{\mu}m$), a specific mitochondrial antioxidants, and cyclosporin A ($1-5{\mu}m$), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam ($1-50{\mu}m$), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.