• Molecular & Cellular Toxicology
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• v.4 no.1
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• pp.16-21
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• 2008

Cell death of trophoblast, particularly by abnormal release of physiological nitric oxide (NO) has been known to be a causative factor of pre-eclampsia. In the present study, effects of intracellular calcium increase enhancing the activity of NO synthases (neuronal NO synthase, nNOS in this trophoblast cells) on the cell death were examined in a human placental full-term cell line (HT-1). Furthermore, we analyzed the possible mechanisms underlying the augmentation of $Ca^{++}$-mediated NOS activity mediated by protein kinases like PKC, PKA, or CaM-KII. In experiments for cell toxicity, a calcium ionophore (ionomycin $10{\mu}M$) enhanced cell death confirmed by MTT assay, and increased significantly nNOS activity determined with a hemoglobin oxidation assay. This cell death was partially protected by pre-treatment of 7-nitroindazole (7-NI, $10{\mu}M$ and $100{\mu}M$), a nNOS-specific inhibitor. Additionally, $Ca^{++}$-ionophore -induced increase of nNOS activity also was partially normalized by pre-treatment of specific inhibitors of protein kinases, PKC, PKA or CaM-KII. Therefore, we suggest that an increase of calcium influx, leading to the activation of nNOS activity, which in turn may result in the death of trophoblast cells by involvement of signaling mechanisms of protein kinases.

• The Korean Journal of Pain
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• v.12 no.2
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• pp.188-190
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• 1999

Background: The goal of this study was to evaluate the effects of gabapentin on postherpetic neuralgia. Gabapentin is a known anti-seizure medication, whose cellular mechanism of action is not well understood. Unlike other anticonvulsant, gabapentin has the advantage of a low toxicity and favorable side effect profile. If has been recently recommended for use in treatment of neuropathic pain. Methods: Twelve patients with a diagnosis of postherpetic neuralgia were prescribed gabapentin after failure of routine therapeutic regimens. The dose of gabapentin ranged 300~1800 mg per day, in three divided doses. If initial dose was ineffective and no side effects were noted, the dosages was increased by 300 mg a day in divided doses, to the maximum level for 2 weeks. Patients were evaluated for analgesia using visual analogue scale (VAS) pain score (0; no pain, 10; worst possible pain) and possible side effects. Results: A significant decrease in pain scores with gabapentin were noted. There were several mild side effects such as dizziness, somnolence, dry mouth, constipation and facial edema, without need of special treatment. Conclusions: Gabapentin may be a useful adjunct for treating intractable postherpetic neuralgia with a minimal side effects.

• The Journal of Pediatrics of Korean Medicine
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• v.29 no.1
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• pp.1-14
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• 2015

Objectives This experimental study was designed to investigate the effects of Dai-saiko-to (DSH) on differentiation of 3T3-L1 preadipocytes and body weight, serum lipid levels in high-fat diet-induced obese mice. Materials and Methods Cells were incubated with DSH at an indicated concentration (0.01-1 mg/ml) for 24h, then the growth rate was assessed by MTS assay. 3T3-L1 preadipocytes were incubated in DMEM for 2 days with the indicated concentrations of DSH. On Day 6, the cells were fixed and the cellular lipid contents were assessed by Oil-Red-O staining. The expression of peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) and cytidine-cytidine-adenosine-adenosine-thymine (CCAAT)/enhancer-binding proteins ${\alpha}$ ($C/EBP{\alpha}$) as adipocyte-specific proteins were determined by real time RT-PCR and western blotting. Four-weeks old mice (wild-type C57BL/6) were used for all experiments. Body weight gain and serum lipid levels were measured in the obesity-induced mice. Results DSH did not show toxicity even at the concentration of 1 mg/ml and DSH significantly inhibited the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. Also, DSH significantly reduced the expressions of $PPAR{\gamma}$ and $C/EBP{\alpha}$ in a dose-dependent manner. Furthermore, DSH significantly reduced body weight gain, serum glucose, total cholesterol and LDL-cholesterol contents in obesity-induced mice. Conclusions These results demonstrated that DSH inhibited 3T3-L1 preadipocyte differentiations and high-fat diet-induced obesity in mice.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.6
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• pp.764-770
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• 2013

Stevia rebaudiana is a traditional herb used as a sweetener in Brazil and Paraguay as well as Korea and China. This study investigated the efficacy of Stevia rebaudiana methanol extract (SRE) to protect cells against the mitochondrial dysfunction and apoptosis in hepatocyte. To determine the effects of SRE on oxidative stress, we used the human derived hepatocyte cell line, HepG2 cell. Treatment of arachidonic acid (AA)+iron in HepG2 cells synergistically amplified cytotoxicity, as indicated by the excess reactive oxygen species (ROS) and mitochondrial permeability transition by fluorescence activated cell sorter (FACS) and immunoblot analysis. Treatment with SRE protected hepatocytes from AA+iron-induced cellular toxicity, as shown by alterations in the protein levels related with cell viability such as procaspase-3. SRE also prevented the mitochondrial dysfunction induced by AA+iron, and showed anti-oxidant effects as inhibition of $H_2O_2$ production and GSH depletion. Moreover, we measured the effects of SRE on AMP-activated protein kinase (AMPK), a key regulator in determining cell survival or death. Acetyl-CoA Carboxylase (ACC), a direct downstream target of AMPK. SRE increased phosphorylation of ACC, and prevented the inhibition of ACC phosphorylation by AA+iron. These results indicated that SRE has the ability to protect cells against AA+iron-induced $H_2O_2$ production and mitochondrial impairment, which may be mediated with AMPK-ACC pathway.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.152-159
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• 2014

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and $PDGFR{\beta}$ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and $PDGFR{\beta}$ fusion proteins were purified by $Ni^{2+}$ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and $PDGFR{\beta}$ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

• Journal of The Korean Society of Grassland and Forage Science
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• v.36 no.3
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• pp.237-242
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• 2016

Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.

• The Journal of Pediatrics of Korean Medicine
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• v.27 no.4
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• pp.17-30
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• 2013

Objectives This experimental study was designed to investigate the effects of Mulberry leaves contained herbal mixture (MLHM) on body weight, serum lipid level and adipocyte differentiation in high fat diet-fed obese mice. Methods Four-week old mice (wild-type C57/BL6) were used for all experiments. Cells were incubated with MLHM at the indicated concentration (0.04-4mg/ml) for 24h, and growth rate was assessed by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3T3-L1 preadipocytes were incubated in DMEM for 2 days with the indicated concentrations of MLHM, and on Day 6, the cells were fixed and the cellular lipid contents were assessed by Oil-Red-O staining. The expression of peroxisome proliferator-activated receptor ${\gamma}$ (PPAR ${\gamma}$) and cytidine-cytidine-adenosine-adenosine-thymine (CCAAT)/enhancer-binding proteins ${\alpha}$ (C/EBP ${\alpha}$) as adipocyte-specific proteins were determined by real time RT-PCR and western blotting. In addition, body weight gain and serum lipid levels were measured in the mice with obesity induced by the high fat-diet for four weeks. Results Though MLHM did not show toxicity even at the concentration of 4mg/ml, MLHM significantly inhibited the differentiation of 3T3-L1 preadipocites in a dose-dependent manner. Also, MLHM significantly reduced the expressions of PPAR ${\gamma}$ and C/EBP ${\alpha}$ in a dose-dependent manner. Furthermore, MLHM significantly reduced body weight gain and LDL-cholesterol contents in high fat diet-fed obese mice. Conclusions These results demonstrate that MLHM exerts anti-obesity effect in 3T3-L1 cells and mice with obesity by high-fat diet.

• Nutrition Research and Practice
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• v.10 no.4
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• pp.393-397
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• 2016

BACKGROUND/OBJECFTIVES: Artemisinin, a natural product isolated from Gaeddongssuk (artemisia annua L.) and its main active derivative, dihydroartemisinin (DHA), have long been used as antimalarial drugs. Recent studies reported that artemisinin is efficacious for curing diseases, including cancers, and for improving the immune system. Many researchers have shown the therapeutic effects of artemisinin on tumors such as breast cancer, liver cancer and kidney cancer, but there is still insufficient data regarding glioblastoma (GBM). Glioblastoma accounts for 12-15% of brain cancer, and the median survival is less than a year, despite medical treatments such as surgery, radiation therapy, and chemotherapy. In this study, we investigated the anti-cancer effects of DHA and transferrin against glioblastoma (glioblastoma multiforme, GBM). MATERIALS/METHODS: This study was performed through in vitro experiments using C6 cells. The toxicity dependence of DHA and transferrin (TF) on time and concentration was analyzed by MTT assay and cell cycle assay. Observations of cellular morphology were recorded with an optical microscope and color digital camera. The anti-cancer mechanism of DHA and TF against GBM were studied by flow cytometry with Annexin V and caspase 3/7. RESULTS: MTT assay revealed that TF enhanced the cytotoxicity of DHA against C6 cells. An Annexin V immune-precipitation assay showed that the percentages of apoptosis of cells treated with TF, DHA alone, DHA in combination with TF, and the control group were $7.15{\pm}4.15%$, $34.3{\pm}5.15%$, $66.42{\pm}5.98%$, and $1.2{\pm}0.15%$, respectively. The results of the Annexin V assay were consistent with those of the MTT assay. DHA induced apoptosis in C6 cells through DNA damage, and TF enhanced the effects of DHA. CONCLUSION: The results of this study demonstrated that DHA, the derivative of the active ingredient in Gaeddongssuk, is effective against GBM, apparently via inhibition of cancer cell proliferation by a pharmacological effect. The role of transferrin as an allosteric activator in the GBM therapeutic efficacy of DHA was also confirmed.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.409-413
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• 2012

Indolicidin (ID) is a 13-residue Trp-rich antimicrobial peptide (AMP) isolated from bovine neutrophils. In addition to having a high antimicrobial potency, it is also toxic to mammalian cells. To develop novel ID-derived AMPs with shorter lengths and enhanced prokaryotic selectivities (meaning potent antimicrobial activity against bacterial cells without toxicity against mammalian cells) over the parental ID, several ID analogs were designed and synthesized. Finally, 10-residue ID analogs (SI, SI-PA, SI-WF and SI-WL) with much higher prokaryotic selectivity than the parental ID were developed. Our results suggest that the hydrophobic and aromatic amino acids at the central position of the analog SI with the highest prokaryotic selectivity are important for potent antimicrobial activity, but two Pro residues do not affect antimicrobial activity. The order of prokaryotic selectivity for ID and its designed analogs was SI > SI-PA > SI-WF > SI-WL > ID > SI-WA. Taken together, our designed short ID analogs could be developed as therapeutic agents for treating bacterial infections.

• Environmental Analysis Health and Toxicology
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• v.11 no.3_4
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• pp.53-57
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• 1996

In this study we investigated the effect of hydroxybrazilin on glutathione depletion induced by BrCCl$_3$ and menadione in cultured hepatocytes to understand the cellular mechanisms of hepatoprotective effect of hydroxybrazilin. Hydroxybrazilin alone had no effect on total glutathione level and the ratio of reduced glutathione/total glutathione (GSH/(GSSG+GSH)). BrCCl$_3$ dramatically decreased total glutathione level and hydroxybrazilin significantly prevented glutathion depletion by BrCCl$_3$. The ratio of GSH/(GSSG+ GSH) was also decreased by BrCCl$_3$ and recovered by hydroxybrazilin treatment. Menadione decreased total glutathione level and the ratio of GSH/(GSSG+GSH) but hydroxybrazilin showed no significant effects on menadione-induced glutathione depletion. These data suggest that hydroxybrazilin might prevent the hepatotoxicity induced by chemicalderived radicals but not the toxicity linked with oxidative stress.