• Tuberculosis and Respiratory Diseases
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• v.66 no.3
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• pp.178-185
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• 2009

Background: Epigallocatechin-3-gallate (EGCG) is the major catechin in green tea, and has shown antiproliferative, antiangiogenic, antimetastatic and cell cycle pertubation activity in various tumor models. Hypoxia can be induced because angiogenesis is insufficient for highly proliferating cancer. Hypoxia-inducible factor-1$\alpha$ (HIF-1$\alpha$) and its downstream target, vascular endothelial growth factor (VEGF), are important for angiogenesis, tumor growth and metastasis. The aim of this study was to determine how hypoxia could cause changes in the cellular phenomena and microenvironment in a non-small cell culture system and to examine the effects of EGCG on a HIF-1$\alpha$ and VEGF in A549 cell line. Methods: A549 cells, a non-small cell lung cancer cell line, were cultured with DMEM and 10% fetal bovine serum. A decrease in oxygen tension was induced using a hypoxia microchamber and a $CO_2-N_2$ gas mixture. Gas analysis and a MTT assay were performed. The A549 cells were treated with EGCG (0, 12.5, 25, 50 ${\mu}mol/L$), and then examined by real-time-PCR analysis of HIF-1$\alpha$, VEGF, and $\beta$-actin mRNA. Results: Hypoxia reduced the proliferation of A549 cells from normoxic conditions. EGCG inhibited HIF-1$\alpha$ transcription in A549 cells in a dose-dependent manner. Compared to HIF-1$\alpha$, VEGF was not inhibited by EGCG. Conclusion: HIF-1$\alpha$ can be inhibited by EGCG. This suggests that targeting HIF-1$\alpha$ with a EGCG treatment may have therapeutic potential in non-small cell lung cancers.

• Clinical and Experimental Pediatrics
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• v.53 no.2
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• pp.178-183
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• 2010

Purpose : The aim of this study was to identify mutations associated with macrolide resistance in Mycoplasma pneumoniae (MP) and to establish a cultural method to determine antimicrobial susceptibility. Methods : Nasopharyngeal aspirates (NPAs) were collected from 62 children diagnosed with MP pneumonia by a serologic method or polymerase chain reaction. The 23S rRNA and L4 ribosomal protein genes of MP were amplified and sequenced. To identify mutations in these 2 genes, their nucleotide sequences were compared to those of the reference strain M129. MP cultivation was carried out for 32 (28 frozen and 5 refrigerated) NPAs and M129 strain using Chanock's glucose broth and agar plate in a 5% $CO_2$ incubator at $37^{\circ}C$ and examined at 2-3 day intervals for 6 weeks. Results : Among the 62 specimens, 17 had M144V mutations in ribosomal protein L4. The A2064G mutation was observed in 1 specimen; its 23S rRNA gene was successfully sequenced. Culture for MP was successful from the M129 strain and 2 of the 5 NPAs that were refrigerated for no longer than 3 days. However, MP did not grow from the 28 NPAs that were kept frozen at $-80^{\circ}C$ since 2003. Conclusion : We found the M144V mutation of L4 protein to be common and that of domain V of 23S rRNA gene was relatively rare among MP. Studies on the prevalence of macrolide-resistant MP and the relationship between the mutations of 23S rRNA gene and ribosomal protein L4 will aid in understanding the mechanism of macrolide resistance in MP.

• Journal of Life Science
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• v.30 no.4
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• pp.359-369
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• 2020

Although many studies on immune modulatory materials have used RAW 264.7 cells, few have used T cell-derived TK-1 cell lines. Moreover, although some studies have investigated the efficacy of plant-derived β-sitosterol, few have examined the immunomodulatory activity of its analogue, daucosterol. In this study, β-sitosterol and daucosterol were isolated from D. batatas and identified by nuclear magnetic resonance spectroscopy. To evaluate the immune-enhancing or inhibitory effects of the isolated phytosterols, the expression levels of the inflammatory response genes COX-2, TNF-α, IL-6, and iNOS were analyzed by RT-PCR. The relative expression levels of TNF-α and iNOS in RAW 264.7 cells were increased more than threefold with β-sitosterol treatment comparing to those of untreated control. In the case of TK-1 cells, the expression level of TNF-α was decreased and the expression level of iNOS was increased in a β-sitosterol concentration-dependent manner. The expression levels of COX-2, TNF-α, and IL-6 increased by approximately 0.7-1.2 times in RAW 264.7 cells treated with daucosterol compared to those of untreated control, but iNOS expression decreased by 0.8-0.18 times. In the case of daucosterol-treated TK-1 cells, the expression levels of TNF-α, IL-6, and iNOS were markedly reduced from those of TK-1 cells treated only with lipopolysaccaride. As a conclusion, β-sitosterol treatment increased TNF-α and iNOS expression levels in RAW 264.7 cells, thus exerting an immune- boosting effect. However, in TK-1 cells, iNOS expression increased while TNF-α expression decreased, indicating an immunosuppressive activity of β-sitosterol. Daucosterol appears to exert an immunosuppressive effect in both macrophages and T cell lines by inhibiting iNOS expression in RAW 264.7 cells and greatly inhibiting the expression of TNF-α, IL-6, and iNOS in TK-1 cells.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.169-175
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• 2005

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin found in grape skins, peanuts, and red wine, has been reported to have a wide range of biological and pharmacological properties. $Amyloid-\beta$ deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease. In this study, we have explored the effects of resveratrol on $amyloid-\beta-peptide-mediated$ cytotoxicity in vitro and modulation of cell growth-regulatory gene products in astroglioma C6 cells to elucidate its possible mechanism for anti-cytotoxicity. Exposure of C6 cells to $Amyloid-\beta$ resulted in dose-dependent growth inhibition and morphological changes of C6 cells, which were recovered by pre-treatment with resveratrol. The anti-proliferative effect of $amyloid-\beta$ was associated with the induction of tumor suppressor p53 and cyclin-dependent kinase (Cdk) inhibitor p21 (WAF1/CIP1) expression assessed by RT-PCR and Western blot analysis in time-dependent manner in C6 cells. In addition, the pro-apoptotic Bax expression was also up-regulated in $amyloid-\beta-treated$ C6 cells without alteration of anti-apoptotic Bcl-2 and $Bcl-X_L$ expression. However, pre-treatment of resveratrol significantly inhibited $amyloid-\beta-induced$ p53, p21 and Bax levels, suggesting that the modulation of p53, p21 and Bax levels could be one of the possible pathways by which resveratrol functions as anti-cytotoxic agent. Our results demonstrate that resveratrol may enhance the protection against $amyloid-\beta-induced$ cytotoxicity by promoting the survival of glial cells.

• Journal of Food Hygiene and Safety
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• v.25 no.2
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• pp.153-161
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• 2010

The purpose of this study was to examine the appearance of norovirus in the water for food in food service institutions and the influence of physicochemical and microbial factors of norovirus in order to work out basic data to predict the detection of norovirus. Among 82 samples of water for food in food service institutions, norovirus appeared in 7 samples and the rate of appearance was 8.5%. As for the type of norovirus, one samples contained GI type (genotype GI-6) and six samples contained GII type (genotype GII-2, GII-4, GII-12). In the regression model of prediction of norovirus, the rate of appearance was correlated with $NH_3$-N, total solids and the consumption of $KMnO_4$, out of such variables as $NH_3$-N, total solids, the consumption of $KMnO_4$, depth, chloride and total colony counts, and its contribution rate for effectiveness was 78.60%. In order to examine the influential factor of environment upon the detection of norovirus, Pearson's correlation analysis was carried out. The predictable regression formula for appearance rate of norovirus was expressed as -1.818 + 42.677 [$NH_3$-N] + 0.023 [total solids] + 0.762 [consumption of $KMnO_4$] -0.009 [depth] -0.146 [chloride] + 0.007 [total colony counts] (R = 0.904, $R^2$ = 0.818, adjusted $R^2$ = 0.786, p < 0.05). The most influential factors upon the detection of norovirus were $NH_3$-N, total solids and the consumption of $KMnO_4$. In other words, when the measured values of $NH_3$-N, total solids and the consumption of $KMnO_4$ were higher, the possibility of appearance of norovirus increased.

• Journal of Life Science
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• v.20 no.1
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• pp.103-112
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• 2010

Pemetrexed has demonstrated clinical activity in non-small cell lung cancer (NSCLC) as well as other solid tumors. It transports into the cells via reduced folate carrier (RFC) and is polyglutamated by folypolyglutamate synthetase (FPGS). Pemetrexed directly inhibits several folate-dependent enzymes such as thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). We investigated the effects of genetic variations and the expression of RFC, FPGS, TS and DHFR enzymes on drug sensitivity to pemetrexed in NSCLC cells. Polymorphisms in RFC, FPGS, and DHFR were genotyped in four NSCLC cells - A549, PC14, HCC-1588, and H226. Real-time RT-PCR and Western blot was performed to evaluate mRNA transcripts and protein of these genes. The cytotoxicity of pemetrexed was measured by SRB assay. In PC14 and H226 cells, increased mRNA expressions of RFC and FPGS were associated with higher cytotoxicity to pemetrexed. 2R/2R genotype of TS and its increased mRNA expression were associated with drug resistance to pemetrexed in A549 cells, whereas 3R/3R genotype in TS with decreased mRNA expression was associated with higher sensitivity in H226 cells. After pemetrexed treatment, an inverse change of DHFR mRNA and protein expression was found. The strongest linkage disequilibrium (LD) was discovered between-1726C>T and -1188A>C SNP of DHFR gene. Our findings suggest the cytotoxic effect of pemetrexed may be associated with genetic polymorphisms and the expression level of genes involved in pemetrexed metabolisms in NSCLC cells.

• Journal of Life Science
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• v.27 no.7
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• pp.739-745
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• 2017

In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy. $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) predominantly converts progesterone into its biologically inactive form $20{\alpha}$-hydroxyprogesterone ($20{\alpha}$-OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In this study, we characterized the expression and localization of $20{\alpha}$-HSDinthe testis of MediKinetics $Micropigs^{(R)}$. The testes were collected at days 6, 9, 12, 18, and 21 after birth. The $20{\alpha}$-HSD mRNA was found to be expressed in the testis at day 6 after birth by RT-PCR. The highest level of mRNA expression in the testis was detected on day 21 after birth. However, the mRNA was not detected in the placenta after parturition. Western blot for $20{\alpha}$-HSD reveal that the specific 37-kDa band was detected in immature pig testis. However, this band was not detected in testis tissue at day 6 after birth. In the immunohistochemical analysis of the testis, $20{\alpha}$-HSD was detected in the Sertoli cells and Leydig cells. Taken together, our study shows for the first time that the $20{\alpha}$-HSD mRNA and protein are expressed in pig testis after birth. Further investigation is required to elucidate the functional mechanisms of $20{\alpha}$-HSD in pig testis after birth.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.43 no.2
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• pp.157-164
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• 2017

Stevia (Stevia rebaudiana) is a perennial plant of the genus Stevia, originated in South America. It stores many forms of glycosides, mainly stevioside and rebaudioside A, in which steviol is the basic structure. Steviol glycosides, widely used as sweeteners, are superior to sugar in sweetness. Recently, it has been reported that steviol glycosides are involved not only in the skin whitening and anti-inflammatory effect but also in enhancing skin barrier function through tight junction regulation. Thus, we examined anti-inflammatory effect of rebaudioside A and tried to identify its potential for improving atopic dermatitis as cosmetic ingredients. To investigate the anti-inflammatory effect, cell viability and mRNA expression level of inflammation-related cytokines were measured using mouse macrophage RAW264.7 cells. Cell counting kit 8 (CCK-8) assay was carried out to measure cell viability and the maximum concentration without cytotoxicity was set to $250{\mu}M$. A quantitative real-time RT-PCR method was used for the study of the inflammatory suppression of rebaudioside A. Rebaudioside A inhibited expression of inducible nitric oxide synthase (iNOS) up to 47% and COX-2 up to 41% compared to LPS treated condition. NO synthesis was decreased by rebaudioside A. Also, mRNA expression of interleukin (IL)-$1{\alpha}$, IL-$1{\beta}$ and IL-6 in LPS-stimulated RAW264.7 cells was decreased to 40%, 45% and 59%, respectively, as a concentration-dependent manner. In conclusion, rebaudioside A inhibited the inflammatory response by regulation of cytokine gene expression. From these results, we expect that steviol glycoside, such as rebaudioside A, can be used as a material for improving atopic dermatitis in the future.

• Food Science and Preservation
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• v.21 no.1
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• pp.97-106
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• 2014

This study was performed in order to investigate the anti-obesity effect of Polygala tenuifolia on lipid mechanism in 3T3-L1 adipocytes. The chemical composition of the P. tenuifolia was analyzed in order to assess its nutritional value. Total dietary fiber was the highest among the proximate component of the P. tenuifolia. These results showed that the P. tenuifolia may be used as a potential functional ingredient for anti-obesity effect. Intracellular lipid droplets in the adipocyte were stained with oil-red O dye and quantified. In comparison to the control, lipid accumulation was significantly decreased by 40.1% and 22.4% when treated with the water extract and 70% EtOH extract of the P. tenuifolia at the concentration of $10{\mu}g/mL$, respectively. The anti-adipogenic effect of the water extract was stronger than that of the 70% EtOH extract. The gene expression levels were measured via Western blot and real-time PCR. As a result, the water extract was found to have decrease the gene expression of SREBP-1c, PPAR, $C/EBP{\alpha}$, FAS, ACC in a dose-dependent manner. These indicate that the water extract inhibits pre-adipocyte differentiation and adipogenesis by blocking the SREBP-1c gene expression in 3T3-L1 cells. Therefore, P. tenuifolia can be used as an effective anti-obesity agent.

• Journal of Life Science
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• v.19 no.11
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• pp.1637-1643
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• 2009

Shikonin, a component of Lithospermum erythrorhizon Sieb. et Zucc, exerts various characteristics such as anti-inflammatory, anti-cancer and anti-obesity functions. To elucidate the molecular mechanism of shikonin-induced inhibition of adipogenesis, we analyzed the mRNA expression level of various adipogenesis-related factors including C/EBPs (CCAAT/enhancerbinding proteins) and $PPAR{\gamma}$ (peroxisome proliferator-activated receptor $\gamma$). The data showed that mRNA expressions of C/$EBP{\beta}$ and C/$EPB{\delta}$ were only slightly changed by shikonin treatment, but mRNA expressions of $PPAR{\gamma}$ and C/$EPB{\alpha}$ were significantly down-regulated. Then, we tested whether upstream regulators of C/$EBP{\beta}$ and $PPAR{\gamma}$ were involved in anti-adipogenesis of shikonin. C/$EBP{\gamma}$ and CHOP (C/EBP homologous protein), which are upstream regulators of C/$EBP{\beta}$, were not affected by shikonin treatment. On the contrary, the mRNA level of KROX20 was markedly down-regulated by shikonin treatment. These results suggest that KROX20 might regulate downstream factors of adipogenesis through C/$EBP{\beta}$-independent pathway. The expression of KLF15 (Kruppel-like factor15), which is a member of KLF family and is a upstream regulator of $PPAR{\gamma}$, was dramatically decreased by shikonin treatment, but KLF2 was not changed. Shikonin had no impact on the expression of KLF5 in the early stage of adipogenesis, but shikonin increased expression of KLF5 in the late stage of adipogenesis. Even though mRNA expression of KLF5 was moderately changed by shikonin treatment, its effect may be small compared with the effect of KLF15, which was markedly inhibited. Taken together, these results suggest that shikonin inhibits adipogenesis through the down-regulation of $PPAR{\gamma}$ and C/$EPB{\alpha}$, which is mediated by the down-regulation of two pro-adipogenic factors, KROX20 and KLF15.