• The Korea Journal of Herbology
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• v.22 no.2
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• pp.147-153
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• 2007

Objectives : Sophora flavescens (SF) is widely used in traditional herbal medicine in Korea and is well recognized for its anti-inflammatory effect. However, its effect on Tumornecrosis factor alpha (Tnf) production in BV2 microglial cell is not yet known. Methods : We investigated the effect of SF on the production and expression of Tnf, a well known inflammatory mediator, in lipopolysaccaride (LPS)-activated BV2 microglial cells. Results : The LPS-induced Tnf production was markedly reduced by treatment with SF (50 ${\mu}g/ml$). In reverse transcription polymerase chain reaction (RT-PCR) analysis, SF suppressed the LPS activated expression of Tnf mRNA. In addition, Western blot analysis confirmed that SF suppressed the expression of Tnf. Sophora flavescens also inhibited the LPS-induced phosphylation of extracellular signal-regulated kinases (ERK), which mediate the Tnfproduction signaling pathway whereas LPS-induced phosphylation of p38 mitogen activated protein kinase (p38 MAPK), and c-Jun NH2-terminal kinases (JNK) was not inhibited by SF, which implies that SF suppresses LPS-induced Tnf production via the ERK mediated pathway. Conclusion : Taken together, these findings indicated that SF inhibits LPS-induce Tnf production, and that this inhibitory effect is mediated via the ERK pathway.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1311-1319
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• 2016

Alternaria brassicicola (Schwein.) invades Brassicaceae and causes black spot disease, significantly lowering productivity. Mitogen-activated protein kinases (MAPKs) and their upstream kinases, including MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKK), comprise one of the most important signaling pathways determining the pathogenicity of diverse plant pathogens. The AbSte7 gene in the genome of A. brassicicola was predicted to be a homolog of yeast Ste7, a MAPKK; therefore, the function was characterized by generating null mutant strains with a gene replacement method. AbSte7 replacement mutants (RMs) had a slower growth rate and altered colony morphology compared with the wild-type strain. Disruption of the AbSte7 gene resulted in defects in conidiation and melanin accumulation. AbSte7 was also involved in the resistance pathways in salt and oxidative stress, working to negatively regulate salt tolerance and positively regulate oxidative stress. Pathogenicity assays revealed that AbSte7 RMs could not infect intact cabbage leaves, but only formed very small lesions in wounded leaves, whereas typical lesions appeared on both intact and wounded leaves inoculated with the wild-type strain. As the first studied MAPKK in A. brassicicola, these data strongly suggest that the AbSte7 gene is an essential element for the growth, development, and pathogenicity of A. brassicicola.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2003.10a
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• pp.175-175
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• 2003

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2003.06a
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• pp.152-153
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• 2003

• Archives of Pharmacal Research
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• v.27 no.4
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• pp.429-435
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• 2004

Ginseng has an anti-cancer effect in several cancer models. This study was to characterize active constituents of ginseng and their effects on proliferation of prostate cancer cell lines, LNCaP and PC3. Cell proliferation was measured by $[^3H]$thymidine incorporation, the intracellular calcium concentration by a dual-wavelength spectrophotometer system, effects on mite-gen-activated protein (MAP) kinases by Western blotting, and cell attachment and morphologic changes were observed under a microscope. Among 11 ginsenosides tested, ginsenosides $Rg_3\;and\;Rh_2$ inhibited the proliferation of prostate cancer cells. $EC_{50}s\;of\;Rg_3\;and\;Rh_2$ on PC3 cells were $8.4{\mu}M\;and\;5.5{\mu}M$, respectively, and $14.1{\mu}M\;and\;4.4{\mu}M$ on LNCaP cells, respectively. Both ginsenosides induced cell detachment and modulated three modules of MAP kinases activities differently in LNCaP and PC3 cells. These results suggest that ginsenosides $Rg_3\;and\;Rh_2$-induced cell detachment and inhibition of the proliferation of prostate cancer cells may be associated with modulation of three modules of MAP kinases.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.71-76
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• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• 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.

• Nutrition Research and Practice
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• v.11 no.5
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• pp.357-364
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• 2017

BACKGROUND/OBJECTIVES: Oxidative stress is closely related with inflammation and development of many diseases. Black soybean seed coat contains high amount of anthocyanins, which are well-known for free radical scavenging activities. This study investigated inflammatory response and action mechanism of black soybean anthocyanins with regard to antioxidant activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. MATERIALS/METHODS: RAW 264.7 cells were treated with anthocyanins extracted from black soybean seed coats in a concentration range of 12.5 to $100{\mu}g/mL$. The production of reactive oxygen species (ROS), secretion of pro-inflammatory mediators and cytokines, and the signaling in the mitogen activated protein kinases (MAPKs) pathway were examined. RESULTS: Black soybean anthocyanins significantly decreased LPS-stimulated production of ROS, inflammatory mediators such as nitric oxide (NO) and prostaglandin $E_2$, and pro-inflammatory cytokines, including tumor necrosis factor ${\alpha}$ and interleukin-6, in a dose-dependent manner without cytotoxicity (P < 0.001). Black soybean anthocyanins downregulated the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells (P < 0.001). Moreover, black soybean anthocyanins inhibited LPS-induced phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 (P < 0.001). CONCLUSION: These results suggest that black soybean anthocyanins exert anti-inflammatory activity by inhibiting ROS generation and subsequent MAPKs signaling, thereby inhibiting inflammatory responses.

• Journal of Ginseng Research
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• v.42 no.1
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• pp.75-80
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• 2018

Background: The aim of the present study was to evaluate the potential protective effects of six ginsenosides (Rb1, Rb2, Rc, Rd, Rg1, and Rg3) isolated from Panax ginseng against tacrolimus (FK506)-induced apoptosis in renal proximal tubular LLC-PK1 cells. Methods: LLC-PK1 cells were treated with FK506 and ginsenosides, and cell viability was measured. Protein expressions of mitogen-activated protein kinases, caspase-3, and kidney injury molecule-1 (KIM-1) were evaluated by Western blotting analyses. The number of apoptotic cells was measured using an image-based cytometric assay. Results: Reduction in cell viability by $60{\mu}M$ FK506 was ameliorated significantly by cotreatment with ginsenosides Rg1 and Rb1. The phosphorylation of p38, extracellular signal-regulated kinases, and KIM-1, and cleavage of caspase-3, increased markedly in LLC-PK1 cells treated with FK506 and significantly decreased after cotreatment with ginsenoside Rb1. The number of apoptotic cells decreased by 6.0% after cotreatment with ginsenoside Rb1 ($10{\mu}M$ and $50{\mu}M$). Conclusion: The antiapoptotic effects of ginsenoside Rb1 on FK506-induced apoptosis were mediated by the inhibition of mitogen-activated protein kinases and caspase activation.

• Biomolecules & Therapeutics
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• v.25 no.6
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• pp.593-598
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• 2017

The $Na^+/H^+$ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular $H^+$ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-${\beta}$ (PKC-${\beta}$) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to $100{\mu}M$ glutamate or 20 mM $NH_4Cl$. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-${\beta}$, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to $NH_4Cl$. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-${\beta}$, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-${\beta}$/ERK1/2/p90RSK pathway in neuronal cells.