• The Journal of Korean Medicine
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• v.25 no.2
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• pp.127-137
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• 2004

Objectives : The purpose of this investigation was to evaluate the effects of Sohaphyang-won (SH) on the alteration in gene expression in a hypoxia model using cultured rat cortical cells. Methods : E18 rat cortical cells were grown in neurobasal medium containing B27 supplement. On 12 DIV, SH was added ($20\mu\textrm{g}/ml$) to the culture media for 24 hrs. On 14 DIV, cells were given a hypoxic insult (2% O2/5% CO2, $37^{\circ}C$, 3 hrs), returned to normoxia and cultured for another 24 hrs. Total RNA was prepared from SH-untreated (control) and -treated cultures and alteration in gene expression was analyzed by microarray using rat 5K-TwinChips. Results : Effects on some of the genes whose functions are implicated in neural viability are as follows: 1) For most of the genes altered in expression, the global M values were between -05 to +0.5, Among these, 1517 genes were increased in their expression by more than global M +0.1, while 1480 genes were decreased by more than global M -0.1. 2) The expression of apoptosis-related genes such as Bad (global M =0.35), tumor protein p53 (T53) (global M =0.28) were increased, while v-akt murine thymoma viral oncogene homolog 1 (Akt1) was decreased. 3) The expression of hemoglobin alpha 1 (probably neuroglobin) was increased by about 3.2-fold (global M =1.7). 4) The expression of antioxidation-related catalase gene was increased (global M =0.26). 5) The expression of PKCzeta (prkcz), an upstream kinase of MAPK, was increased (global M =0.29). 6) The expression of retinoic acid receptor alpha (RAR), which may regulate transcription in hypoxic stress, was increased (global M =10.27). Conclusions : In summary, the microarray data suggest that SH doesn't increase the expression of oxygen capture-, anti-oxidation- and 'response to stress' -related genes but decreases some anti-apoptosis genes which would help protect the hypoxic cells from apoptosis.

• Molecules and Cells
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• v.39 no.3
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• pp.195-203
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• 2016

Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions ($Cu^+$ and $Cu^{2+}$), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped $Cu_2O$ and CuO crystals were prepared to test the role of the two different ions, $Cu^+$ and $Cu^{2+}$, respectively. The $Cu_2O$ crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The $Cu_2O$ crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. $Cu_2O$ crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit $Cu_2O$-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of $Cu^+$ ions in the vascular system, where $Cu^+$ induces autophagy while $Cu^{2+}$ has no detected effect.

• Toxicological Research
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• v.35 no.4
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• pp.361-369
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• 2019

1,2-Dichloropropane (1,2-DCP) has been used as an industrial solvent and a chemical intermediate, as well as in soil fumigants. Human exposure may occur during its production and industrial use. The target organs of 1,2-DCP are the eyes, respiratory system, liver, kidneys, central nervous system, and skin. Repeated or prolonged contact may cause skin sensitization. In this study, 1,2-DCP was dissolved in corn oil at 0, 2.73, 5.75, and 8.75 mL/kg. The skin of mice treated with 1,2-DCP was investigated using western blotting, hematoxylin and eosin staining, and immunohistochemistry. 1,2-DCP was applied to the dorsal skin and both ears of C57BL/6J mice. The thickness of ears and the epidermis increased significantly following treatment, and the appearance of blood vessels was observed in the dorsal skin. Additionally, the expression of vascular endothelial growth factor, which is tightly associated with neovascularization, increased significantly. The levels of protein kinase-B (PKB), phosphorylated PKB, mammalian target of rapamycin (mTOR), and phosphorylated mTOR, all of which are key components of the phosphoinositide 3-kinase/PKB/mTOR signaling pathway, were also enhanced. Taken together, 1,2-DCP induced angiogenesis in dermatitis through the PI3K/PKB/mTOR pathway in the skin.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.7
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• pp.3293-3297
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• 2014

Background: Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. Methods: In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Results: Further experiments showed that ${\alpha}$-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Conclusion: Taken together, these findings indicate that ${\alpha}$-pinene may be useful as a potential anti-tumor drug.

• Journal of Life Science
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• v.19 no.4
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• pp.520-525
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• 2009

Early onset of Batten disease (EBD), one of the most lethal neurodegenerative storage disorders of childhood, is caused by inactivating mutations in the Ceroid Lipofuscinosis, Neuronal (CLN1) gene. Neurogranin, a calmodulin-binding protein, is expressed in the brain and participates in the protein kinase C (PKC) signaling pathway. While oxidative stress is the suggested cause of neurodegeneration in EBD, its molecular mechanism(s) remains obscure. In this research, we examined the levels of neurogranin in the brain mRNA of wild-type (WT) mice and EBD knockout (KO) mice, as well as the proteins. We also performed neuronal cultures to measure the expression levels of neurgranin and phosphorylated-neurogranin with or without oxidative stress inducers and anti-oxidants. Results showed that neurogranin in both EBD KO mice brain mRNA and protein extracts decreased in an age dependent manner. However, high amounts of phosphorylated-neurogranin were detected in the 6-month brain. This pattern was also confirmed by cultured neurospheres samples. Moreover, neurospheres treated with $H_2O_2$, an oxidative stress inducer, showed increased phosphorylated-neurogranin patterns. Interestingly, this pattern returned to normal status when treated with N-acetyl-L-cystein, an anti-oxidant, after $H_2O_2$ treatment was performed. Our results suggest that the phosphorylation of neurogranin is affected by oxidative stress status in EBD, and appropriate anti-oxidant treatment will relieve hyper-phosphorylation of neurogranin.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.379-383
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• 2009

Nitric oxide (NO), a diffusible gas, is produced in the central nervous system, including the spinal cord dorsal horn and the trigeminal nucleus, the first central areas processing nociceptive information from periphery. In the spinal cord, it has been demonstrated that NO acts as pronociceptive or antinociceptive mediators, apparently in a concentration-dependent manner. However, the central role of NO in the trigeminal nucleus remains uncertain in support of processing the orofacial nociception. Thus, we here investigated the central role of NO in formalin (3%)-induced orofacial pain in rats by administering membrane-permeable or -impermeable inhibitors, relating to the NO signaling pathways, into intracisternal space. The intracisternal pretreatments with the NO synthase inhibitor L-NAME, the NO-sensitive guanylate cyclase inhibitor ODQ, and the protein kinase C inhibitor GF109203X, all of which are permeable to the cell membrane, significantly reduced the formalin-induced pain, whereas the membrane-impermeable NO scavenger PTIO significantly enhanced it, compared to vehicle controls. These data suggest that an overall effect of NO production in the trigeminal nucleus is pronociceptive, but NO extracellularly diffused out of its producing neurons would have an antinociceptive action.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.79-79
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• 2003

Recently diabetes has been found to be associated with metabolic bone diseases such as osteoporosis. In the present study, attempts have been made-to explore the effect of high glucose in bone formation. Osteoblast-like UMR 106 cells were treated with high glucose (22mM, 33mM, 44mM) for 1 or 2 days. High glucose significantly inhibited proliferation of UMR106 cells in a time- and dose- dependent manner as evidenced by MTT assay. For the evaluation of collagen synthesis, UMR 106 cells were cultured in high glucose media (44mM) for 24 h and the ratio of collagen content to total protein was measured. In addition, gene expression pattern of type I collagen was assessed by RT-PCR. The high concentration of glucose inhibited a collagen synthesis, a marker of bone formation activity. JNK, c- Jun N-terminal Kinase, is known to play an important role in stress-associated cell death. In this regard, we tested to determine whether high glucose has any effect on JNK activity. It has been found that treatment of high glucose induced phosphorylation of JNK. On the other hand, ERK phosphorylation was inhibited by high glucose in a dose-dependent manner. Taken together, Therefore these results indicate that inhibition of proliferation in UMR 106 cells following high glucose is related to JNK/ERK containing signal pathways. This study showed high glucose concentration could alter the bone metabolism leading to defective bone formation, suggesting that high glucose due to diabetes may playa significant role in the development of metabolic bone disease.

• Molecules and Cells
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• v.39 no.4
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• pp.322-329
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• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

• Biomolecules & Therapeutics
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• v.18 no.1
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• pp.32-38
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• 2010

Activation of JNK has long been associated with the apoptotic response induced by various anti-cancer drugs including doxorubicin, vinblastine, and etoposide. In this study, we examined and compared patterns of apoptosis and JNK activation according to three different anti-cancer drugs (daunorubicin, vinblastine, and etoposide) and two different sources of HL60 cells (Jackson Laboratory and ATCC). HL60 cells from Jackson Laboratory (HL60/RPMI) were maintained in RPMI 1640 containing 5% fetal bovine serum and those from ATCC (HL60/IMDM) in IMDM containing 20% fetal bovine serum as to each manufacture's guideline. In general, HL60/RPMI cells were more sensitive to anti-cancer drugs compared to HL60/IMDM cells, demonstrated by the XTT and flow cytometric analyses. Apoptotic pathways after treatment with anti-cancer drugs seemed to be different between HL60/RPMI (daunorubicin and etoposide, caspase 3 dependent, but caspase 8 or 9 independent; vinblastine, caspase 3 independent) and HL60/IMDM (caspase 3 and caspase 9 dependent). The expression of apoptotic protein, BID, was consistent with caspase 3 activation. Immunoblotting of phospho-JNK and JNK kinase assay showed JNK activation by all three anti-cancer drugs in HL60/RPMI, while JNK activation was observed only in vinblastine-treated cells in HL60/IMDM. Our study results suggest that in vitro environmental conditions have a significant influence on JNK mediated apoptosis of HL60 cells by anti-cancer drugs and in vitro culture conditions are important factors in JNK or possibly other MAPK related studies.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.3
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• pp.279-286
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• 2017

The root bark extract of Aralia taibaiensis is used traditionally for the treatment of diabetes mellitus in China. The total saponin extracted from Aralia Taibaiensis (sAT) has effective combined antihyperglycemic and hypolipidemic activities in experimental type 2 diabetic rats. However, the active compounds have not yet been fully investigated. In the present study, we examined effects of twelve triterpenoid saponins on AMP-activated protein kinase (AMPK) activation, and found that compound 28-O-${\beta}$-D-glucopyranosyl ester (AT12) significantly increased phosphorylation of AMPK and Acetyl-CoA carboxylase (ACC). AT12 effectively decreased blood glucose, triglyceride (TG), free fatty acid (FFA) and low density lipoprotein-cholesterol (LDL-C) levels in the rat model of type 2 diabetes mellitus (T2DM). The mechanism by which AT12 activated AMPK was subsequently investigated. Intracellular ATP level and oxygen consumption were significantly reduced by AT12 treatment. The findings suggested AT12 was a novel AMPK activator, and could be useful for the treatment of metabolic diseases.