• Title/Summary/Keyword: IS-PCR

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Stimulating effect of modified Goa-Gi-Um herbal remedy on melanogenesis in B16F10 melanoma cells (B16F10 멜라노마세포에서 과기음가미방의 멜라닌 생성 촉진 효과)

  • Moon, Na-Rang;Kim, Se Yoon;Lee, Jin Hyuk;Lee, Jung Bok;Park, Sunmin
    • The Korea Journal of Herbology
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    • v.28 no.3
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    • pp.69-74
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    • 2013
  • Objectives : Since hypopigmentation is known to increase the risk of skin cancer, melanogenesis in the skin needs to be regulated. Here, we evaluated the melanogenesis stimulatory effects of a modified Goagium herbal remedy (HR) and HR+ox bile (Bos taurus domesticus) extract (OBE) to address hypopigmentation disorders. Methods : B16F10 melanoma cells were treated with different dosages of HR and HR+OBE for 24 to 48 h after 1 h of 10 nM ${\alpha}$-melanocyte stimulating hormone (${\alpha}$-MSH). After the treatment, cell viability, tyrosinase activity, melanin synthesis and the expression of genes related to melanin synthesis were measured and the regulation of the ${\alpha}$-MSH signalling through cAMP responding element binding protein (CREB) was determined. Results : HR and HR+OBE with the ranges of $15{\sim}100{\mu}g/mL$ did not affect cell viability in melanoma cells. The 1 h treatment of HR+OBE (50 and $100{\mu}g/mL$) potentiated the phosphorylation of CREB by enhancing ${\alpha}$-MSH signaling and its 24 h treatment increased CREB expression. Consistent with CREB potentiation, their treatment for 24 h, the expression of microphthalmia-associated transcription factor (MIFT), tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 were increased in realtime PCR. Ultimately, the 48 h treatment of HR+OBE (50 and $100{\mu}g/mL$) increased tyrosniase activity and melanin contents in the melanoma cells in comparison to the control. Conclusions : HR+OBE (50 and $100{\mu}g/mL$) increases melanin synthesis in B16F10 melanoma cells via the stimulation of tyrosinase activity and expression of MIFT, tyrosinase, TRP-1 and TRP-2. HR+OBE can be used as the a possible treatment for hypopigmentation of the skin.

Hepatoprotecive Effects of Puerariae Flos Extract on Experimental Non-alcoholic Fatty Liver Disease Models (비알콜성 지방간 동물모델에서 갈화추출물의 간보호 효과)

  • Hwang, Hyeong-Chil;Kong, Ryong;Kang, Ok-Hwa;Kwo, Dong-Yeul
    • The Korea Journal of Herbology
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    • v.33 no.5
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    • pp.1-8
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    • 2018
  • Objectives : The aim of this study is to investigate the preventive effect of Puerariae Flos ethanol extract (PE) on methionin and choline deficient (MCD)-diet-induced Nonalcoholic fatty liver disease (NAFLD) in C57BL/6J mice. Methods : In the in vivo experiments, C57BL/6J mice were divided into 4 groups; Normal group, Control group, MCD+PE 100 group, and MCD+PE 300 group. After 4 weeks, body weight, liver weight, biochemical parameters for liver function test, histological changes, reverse transcription polymerase chain reaction (RT-PCR), and Western blot were assessed. Results : Mice lost body weight with the MCD-diet and the MCD+PE 100 group and MCD+PE 300 groups lost less than the control group, though showed no statistical significance. Liver weights were decreased by the MCD diet, but MCD+PE 300 groups were increased significantly. In the liver function test, all the values were decreased with the MCD-diet, MCD+PE 100 group and MCD+PE 300 groups were increased significance. In histological findings of the livers, MCD-diet induced severe fatty accumulation in the livers, but this fatty change was reduced in the MCD+PE 100 group and MCD+PE 300 groups was inhibited respectively. In lipid accumulation factors (such as SREBP-1c, $C/EBP{\alpha}$, PPAR-${\gamma}$), MCD+PE 100 group and MCD+PE 300 groups showed inhibitory effect on liver lipogenesis by reducing associated gene expressions caused by MCD diet. Conclusions : We were able to know that Puerariae Flos ethanol extract (PE) shown hepatoprotective effects via a decrease on the hepatic lipogenesis factors in the experimental NAFLD Models.

Efficacy of Alismatis Orientale Rhizoma on Obesity induced by High Fat Diet (고지방식이로 유발된 비만에 대한 택사의 항비만 효과)

  • Jeong, Hyang Sook
    • The Korea Journal of Herbology
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    • v.28 no.3
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    • pp.95-106
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    • 2013
  • Objectives : The researcher investigated the anti-obesity effect of Alismatis Orientale Rhizoma(AP) water extract in mice fed a high fat diet and focused on the analysis of local area adipose tissue. Methods : Male ICR mice were divided into three groups, which were fed either a normal AIN diet, a 45% high fat diet (CT group), or a high fat diet and orally administration with a concentrations of 100 mg/kg (AP100 group) and 300 mg/kg body weight (AP300 group) for eight weeks. Results : As compared with CT group, AP100 group showed significant reductions in absolute weight of liver. As compared with CT group, AP100 group and AP300 group showed significant reductions in weight gain and relative weight of total fat. AST, triglyceride, total-cholesterol levels from the AP 100 group and 300 group were significantly lower than those of the CT, and ALT, LDL-cholesterol levels from the AP 100 group was significantly lower than those of the CT. But serum HDL-cholesterol levels from the AP 100 group and 300 group were significantly higher than those of the CT. And serum adiponectin levels from the AP 100 group was significantly higher than those of the CT. In result of real time PCR, all mRNA expression(PEPCK, G6Pase, PGC-$1{\alpha}$ and $ERR{\gamma}$) of two experimental groups were significantly decreased compared to those of CT group. The treatment with AP on local abdominal area made a fat cell size lessen on the fat tissue in the abdominal cavity and subcutaneous area. Conclusions : These results suggest that AP has an anti-obesity effect and the effect is mediated by inhibition of fat gain.

The Effects of steam heat processing of Helianthus tuberosi Rhizoma on Blood glucose lowering (국우(菊芋) 증자가 혈당강하작용에 미치는 영향)

  • Kim, Jin-Woo;Ha, Mi-Ae;Shin, Yong-Wook
    • The Korea Journal of Herbology
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    • v.32 no.5
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    • pp.39-46
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    • 2017
  • Objective : This study was designed to evaluate the hypoglycemic effects of Helianthus tuberosi Rhizoma extracts and its optimum Heat processing conditions Methods : We investigated the Salivary ${\alpha}$-amylase, pancreas ${\alpha}$-amylase and ${\alpha}$-glucosidase inhibitory activities of extracts from Steam Heated Helianthus tuberosi Rhizoma Ext. The inhibitory activities of a 50% EtOH extract of Steam Heated Helianthus tuberosi Rhizoma Ext against ${\alpha}$-glucosidases were evaluated in this study. Inhibiting these enzymes involved in the absorption of disaccharides significantly decreases the postprandial increase in blood glucose level after a mixed carbohydrate diet. Furthermore, the postprandial blood glucose lowering effect of Steam Heated Helianthus tuberosi Rhizoma Ext. was compared to a known type 2 diabetes drug(Acarbose(R)) in a mice model. Steam Heated Helianthus tuberosus L. Ext significantly reduced the blood glucose increase after glucose loading. Results : The results were confirmed by real-time PCR that after treated with Streptozotocin in L6 cells, induced expression of GLUT4, after the steamed Helianthus tuberosus L. Ext. treated, observed its expression was increased. Steam Heated Helianthus tuberosus L Ext treated 4 hours in L6 cells, cytotoxicity was measured in MTT assay. Its toxicity were 5.7%, 9% and 11.3% at the treatment concentration $12.5{\mu}g/m{\ell}$, $25{\mu}g/m{\ell}$, the $50{\mu}g/m{\ell}$ respectively. Conclusions : Overall, the results of this study indicate that Hypoglycemic effect of Helianthus tuberosi Rhizoma caused by the Steam heat treatment, the optimum Heat processing condition is steamming at $121^{\circ}C$ for 30 min, and it will provide the basis for developing a useful dietary supplement for controlling postprandial hyperglycemia.

High fat diet-induced brain damaging effects through autophagy-mediated senescence, inflammation and apoptosis mitigated by ginsenoside F1-enhanced mixture

  • Hou, Jingang;Jeon, Byeongmin;Baek, Jongin;Yun, Yeejin;Kim, Daeun;Chang, Boyoon;Kim, Sungyeon;Kim, Sunchang
    • Journal of Ginseng Research
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    • v.46 no.1
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    • pp.79-90
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    • 2022
  • Background: Herbal medicines are popular approaches to capably prevent and treat obesity and its related diseases. Excessive exposure to dietary lipids causes oxidative stress and inflammation, which possibly induces cellular senescence and contribute the damaging effects in brain. The potential roles of selective enhanced ginsenoside in regulating high fat diet (HFD)-induced brain damage remain unknown. Methods: The protection function of Ginsenoside F1-enhanced mixture (SGB121) was evaluated by in vivo and in vitro experiments. Human primary astrocytes and SH-SY5Y cells were treated with palmitic acid conjugated Bovine Serum Albumin, and the effects of SGB121 were determined by MTT and lipid uptake assays. For in vivo tests, C57BL/6J mice were fed with high fat diet for 3 months with or without SGB121 administration. Thereafter, immunohistochemistry, western blot, PCR and ELISA assays were conducted with brain tissues. Results and conclusion: SGB121 selectively suppressed HFD-induced oxidative stress and cellular senescence in brain, and reduced subsequent inflammation responses manifested by abrogated secretion of IL-6, IL-1β and TNFα via NF-κB signaling pathway. Interestingly, SGB121 protects against HFD-induced damage by improving mitophagy and endoplasmic reticulum-stress associated autophagy flux and inhibiting apoptosis. In addition, SGB121 regulates lipid uptake and accumulation by FATP4 and PPARα. SGB121 significantly abates excessively phosphorylated tau protein in the cortex and GFAP activation in corpus callosum. Together, our results suggest that SGB121 is able to favor the resistance of brain to HFD-induced damage, therefore provide explicit evidence of the potential to be a functional food.

LC-MS/MS-based Proteomic Analysis of Locally Advanced Rectal Tumors to Identify Biomarkers for Predicting Tumor Response to Neoadjuvant Chemoradiotherapy

  • Kim, Kyung-Ok;Duong, Van-An;Han, Na-Young;Park, Jong-Moon;Kim, Jung Ho;Lee, Hookeun;Baek, Jeong-Heum
    • Mass Spectrometry Letters
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    • v.13 no.3
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    • pp.84-94
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    • 2022
  • Neoadjuvant chemoradiotherapy (nCRT) is a standard therapy used for locally advanced rectal cancer prior to surgery, which can more effectively reduce the locoregional recurrence rate and radiation toxicity compared to postoperative chemoradiotherapy. The response of patients to nCRT varies, and thus, robust biomarkers for predicting a pathological complete response are necessary. This study aimed to identify possible biomarkers involved in the complete response/non-response of rectal cancer patients to nCRT. Comparative proteomic analysis was performed on rectal tissue samples before and after nCRT. Proteins were extracted for label-free proteomic analysis. Western blot and real-time PCR were performed using rectal cancer cell line SNU-503 and radiation-resistant rectal cancer cell line SNU-503R80Gy. A total of 135 up- and 93 down-regulated proteins were identified in the complete response group. Six possible biomarkers were selected to evaluate the expression of proteins and mRNA in SNU-503 and SNU-503R80Gy cell lines. Lyso-phosphatidylcholine acyltransferase 2, annexin A13, aldo-ketose reductase family 1 member B1, and cathelicidin antimicrobial peptide appeared to be potential biomarkers for predicting a pathological complete response to nCRT. This study identified differentially expressed proteins and some potential biomarkers in the complete response group, which would be further validated in future studies.

Inhibitory Effects of GGX on Lung Injury of Chronic Obstructive Lung Disease (COPD) Mice Model (만성폐쇄성폐질환 동물모델에서 GGX의 폐손상 억제 효과)

  • Kim, Tae Hyeon;Yang, Won Kyung;Lee, Su Won;Kim, Seung Hyung;Lyu, Yee Ran;Park, Yang Chun
    • The Journal of Korean Medicine
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    • v.42 no.3
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    • pp.56-71
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    • 2021
  • Objectives: This study is aimed to evaluate the protective effects of GGX on lung injury of Chronic Obstructive Lung Disease (COPD) mice model. Materials and Methods: C57BL/6 mice were challenged with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) and then treated with vehicle only (Control group), dexamethasone 3 mg/kg (Dexa group), gam-gil-tang 200 mg/kg (GGT group), GGX 100, 200, and 400 mg/kg (GGX group). After sacrifice, its bronchoalveolar lavage fluid (BALF) or lung tissue was analyzed with cytospin, Enzyme-Linked Immunosorbent Assay (ELISA), real-time polymerase chain reaction (PCR) and hematoxylin & eosin (H&E), and Masson's trichrome staining. Results: In the COPD model, GGX significantly inhibited the increase of neutrophils, TNF-𝛼, IL-17A, CXCL-1, MIP2 in BALF and TNF-𝛼, IL-1𝛽, IL-10 mRNA expression in lung tissue. It also decreased the severity of histological lung injury. Conclusion: This study suggests the usability of GGX for COPD patients by controlling lung tissue injury.

Evaluation of an Appropriate Replacement Cycle for Copper Antibacterial Film to Prevent Secondary Infection

  • Je, Min-A;Park, Heechul;Kim, Junseong;Lee, Eun Ju;Jung, Minju;Kim, Minji;Jeong, Mingyoung;Yun, Jiyun;Sin, Hayeon;Jin, Hyunwoo;Lee, Kyung Eun;Kim, Jungho
    • Biomedical Science Letters
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    • v.28 no.3
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    • pp.195-199
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    • 2022
  • The use of copper antibacterial films as an effective infection prevention method is increasing owing to its ability to reduce the risk of pathogen transmission. In this study, we evaluated the bacterial contamination of the antibacterial copper membrane attached to a door handle at a university over time. Six mounting locations with high floating population were selected. In three sites, the door handles with the antibacterial film were exposed, while the remaining three were not attached with the antibacterial films. On days 7 and 14, isolated bacterial strains were inoculated in BHI broth and agar, respectively. Colony-forming units (CFU) were determined after incubation. Strain identification was performed using bacterial 16s rRNA PCR and sequencing. Results showed that the bacterial population on day 14 significantly increased from 6 × 109 CFU/mL (day 7) to 2 × 1010 CFU/mL. Furthermore, strain distribution was not different between the on and off the copper antibacterial film groups. In conclusion, although copper has an antibacterial activity, microbial contamination may occur with prolonged use.

Activating transcription factor 4 aggravates angiotensin II-induced cell dysfunction in human vascular aortic smooth muscle cells via transcriptionally activating fibroblast growth factor 21

  • Tao, Ke;Li, Ming;Gu, Xuefeng;Wang, Ming;Qian, Tianwei;Hu, Lijun;Li, Jiang
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.5
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    • pp.347-355
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    • 2022
  • Abdominal aortic aneurysm (AAA) is a life-threatening disorder worldwide. Fibroblast growth factor 21 (FGF21) was shown to display a high level in the plasma of patients with AAA; however, its detailed functions underlying AAA pathogenesis are unclear. An in vitro AAA model was established in human aortic vascular smooth muscle cells (HASMCs) by angiotensin II (Ang-II) stimulation. Cell counting kit-8, wound healing, and Transwell assays were utilized for measuring cell proliferation and migration. RT-qPCR was used for detecting mRNA expression of FGF21 and activating transcription factor 4 (ATF4). Western blotting was utilized for assessing protein levels of FGF21, ATF4, and markers for the contractile phenotype of HASMCs. ChIP and luciferase reporter assays were implemented for identifying the binding relation between AFT4 and FGF21 promoters. FGF21 and ATF4 were both upregulated in Ang-II-treated HASMCs. Knocking down FGF21 attenuated Ang-II-induced proliferation, migration, and phenotype switch of HASMCs. ATF4 activated FGF21 transcription by binding to its promoter. FGF21 overexpression reversed AFT4 silencing-mediated inhibition of cell proliferation, migration, and phenotype switch. ATF4 transcriptionally upregulates FGF21 to promote the proliferation, migration, and phenotype switch of Ang-II-treated HASMCs.

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose

  • Jonghyeok Shin;Seungjoo Kim;Wonbeom Park;Kyoung Chan Jin;Sun-Ki Kim;Dae-Hyuk Kweon
    • Journal of Microbiology and Biotechnology
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    • v.32 no.11
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    • pp.1471-1478
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    • 2022
  • 2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.