• Journal of Dairy Science and Biotechnology
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• 제36권4호
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• pp.208-219
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• 2018

낙농업 및 유가공 제품의 생산과 유통에서 살모넬라 감염에 의한 살모넬라증의 발생은 빈번하며, 이 세균의 항미생물 제제에 대한 내성 증가 현상 또한 지속되고 있어 새로운 항미생물 제제의 수요는 감소하지 않는다. 세균막의 훼손은 세균 생존을 쉽게 위협할 수 있기 때문에 개발되는 항미생물 제제들은 주로 세균의 막을 표적으로 삼지만, 개발되는 제제들이 실제로 세균막의 훼손을 초래하는지 구별하는 것은 많은 노력과 비용을 수반한다. 본 연구에서는 E. coli 세포막 스트레스에 의해 발현이 유도되고, 세균막 외부공간에서만 위치하며, 그 구조상 많은 단백질의 구조 안정화에 기여할 것으로 예상되는 chaperone 단백질 Spy(spheroplast protein Y)의 유전자에 상응하는 살모넬라 spy 유전자에 gfp(green fluorescence protein) 오페론 융합체를 제조하여, 이 융합체가 Salmonella enterica의 두 혈청형 Enteritidis와 Gallinarum의 세포막 스트레스를 인지하여 GFP 발현량이 크게 증가하는 것을 확인하였다. 또한 세균막 스트레스 신호를 특이적으로 인지하는 이인자 신호전달 체계(two component signal transduction system)인 Bae와 Cpx들이 두 살모넬라 혈청형의 spy 유전자 전사 유도에 필수적임을 확인하였다. 따라서 본 연구에서 사용한 spy-gfp 오페론 융합체는 S. Enteritidis와 S. Gallinarum의 세포막 훼손을 특이적이고 신속하게 인식하는 biosensor로서 활용될 수 있을 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1299-1309
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• 2019

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazine-producing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1-carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the $PA{\Delta}phz1$ mutant and the $PA{\Delta}phz2$ mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant $PA{\Delta}lasR::lacZ$, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

• 생명과학회지
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• 제29권8호
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• pp.871-878
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• 2019

본 연구에서는 Rhododendron brachycarpum (RB, 홍만병초)의 80% 메탄올 추출물 및 분획물의 항암 활성을 규명하고자 하였다. RB n-hexane 분획물은 HT-29 세포에서 가장 높은 활성 저해를 보였다($IC_{50}=20.2{\pm}1.2{\mu}g/ml$). 더욱이, 콜로니와 구형 형성은 수와 크기는 유의적으로 감소시켰다. RB의 n-hexane 분획물에서($0.22{\pm}0.02$ fold change) TOP / FOP 플래시 리포터 억제 활성은 추출물 및 다른 분획물 보다 낮게 나타났다. n-hexane 및 ethyl acetate 분획물은 세포 내 ${\beta}-catenin$의 발현을 조절하였다. 2 차 대사 산물이 ${\beta}-catenin$ 분해를 감소시킬 수 있는지 여부를 조사하기 위해 Western blot을 실시한 결과 n-hexane 분획물에서 $p-GSK3{\beta}$를 조절하였으며, 세포내 ${\beta}-catenin$은 핵에서 정량적인 변화를 가져왔다. 이러한 결과는 RB의 n-hexane 분획물로부터 천연 항암 물질을 포함하고 있음을 보여줍니다.

• 한국수정란이식학회지
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• 제33권4호
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.171-179
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• 2019

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of $p27^{Kip1}$, a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while $p27^{Kip1}$ expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of $p27^{Kip1}$ and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on $p27^{Kip1}$ expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and $p27^{Kip1}$-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and $p27^{Kip1}$ expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and $p27^{Kip1}$ expression. In contrast, miR-186 expression positively associated with $p27^{Kip1}$ expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through $p27^{Kip1}$-mediated cell cycle alternation.

• Journal of Microbiology and Biotechnology
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• 제31권7호
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• pp.933-941
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• 2021

Ginsenoside Rg4 is a rare ginsenoside that is naturally found in ginseng, and exhibits a wide range of biological activities including antioxidant and anti-inflammatory properties in several cell types. The purpose of this study was to use an in vivo model of hair follicle (HF)-mimic based on a human dermal papilla (DP) spheroid system prepared by three-dimensional (3D) culture and to investigate the effect of Rg4 on the hair-inductive properties of DP cells. Treatment of the DP spheroids with Rg4 (20 to 50 ㎍/ml) significantly increased the viability and size of the DP spheres in a dose-dependent manner. Rg4 also increased the mRNA and protein expression of DP signature genes that are related to hair growth including ALP, BMP2, and VCAN in the DP spheres. Analysis of the signaling molecules and luciferase reporter assays further revealed that Rg4 induces the activation of phosphoinositide 3-kinase (PI3K)/AKT and the inhibitory phosphorylation of GSK3β, which activates the WNT/β-catenin signaling pathway. These results correlated with not only the increased nuclear translocation of β-catenin following the treatment of the DP spheres with Rg4 but also the significant elevation of mRNA expression of the downstream target genes of the WNT/β-catenin pathway including WNT5A, β-catenin, and LEF1. In conclusion, these results demonstrated that ginsenoside Rg4 promotes the hair-inductive properties of DP cells by activating the AKT/GSK3β/β-catenin signaling pathway in DP spheres, suggesting that Rg4 could be a potential natural therapy for hair growth.

• Animal Bioscience
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• 제34권2호
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• pp.172-184
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• 2021

Objective: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating VNN1 gene expression in chicken liver. Methods: 5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken VNN1 and miRanda was used to search miRNA binding sites in 3' untranslated region (3'UTR) of chicken VNN1. We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p. Results: Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α (HNF1α), PPARα, and CCAAT/enhancer binding protein α. GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of VNN1 gene expression. In addition, the VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of VNN1 mRNA. Conclusion: This study demonstrates that PPARα is an important transcriptional activator of VNN1 gene expression and that miRNA-181a-5p acts as a negative regulator of VNN1 expression in chicken hepatocytes.

• Yonsei Medical Journal
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• 제59권9호
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• pp.1096-1106
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• 2018

Purpose: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. Materials and Methods: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. $PPAR-{\gamma}$ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and $NF-{\kappa}B$ activity was determined by a Caspase 3 Activity Assay Kit or $NF-{\kappa}B$ p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and $PPAR-{\gamma}$ 3'UTR. Results: MiR-128 expression was upregulated and $PPAR-{\gamma}$ expression was downregulated in plasma from AD patients and $amyloid-{\beta}$ $(A{\beta})-treated$ primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased $A{\beta}-mediated$ cytotoxicity through inactivation of $NF-{\kappa}B$ in MCN and N2a cells. Moreover, $PPAR-{\gamma}$ was a target of miR-128. $PPAR-{\gamma}$ upregulation attenuated $A{\beta}-mediated$ cytotoxicity by inactivating $NF-{\kappa}B$ in MCN and N2a cells. Furthermore, $PPAR-{\gamma}$ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and $NF-{\kappa}B$ activity in MCN and N2a cells. Conclusion: MiR-128 inhibitor decreased $A{\beta}-mediated$ cytotoxicity by upregulating $PPAR-{\gamma}$ via inactivation of $NF-{\kappa}B$ in MCN and N2a cells, providing a new potential target in AD treatment.

• Biomolecules & Therapeutics
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• 제29권2호
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• pp.211-219
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• 2021

Alopecia is a distressing condition caused by the dysregulation of anagen, catagen, and telogen in the hair cycle. Dermal papilla cells (DPCs) regulate the hair cycle and play important roles in hair growth and regeneration. Myristoleic acid (MA) increases Wnt reporter activity in DPCs. However, the action mechanisms of MA on the stimulation of anagen signaling in DPCs is not known. In this study, we evaluated the effects of MA on anagen-activating signaling pathways in DPCs. MA significantly increased DPC proliferation and stimulated the G2/M phase, accompanied by increasing cyclin A, Cdc2, and cyclin B1. To elucidate the mechanism by which MA promotes DPC proliferation, we evaluated the effect of MA on autophagy and intracellular pathways. MA induced autophagosome formation by decreasing the levels of the phospho-mammalian target of rapamycin (phospho-mTOR) and increasing autophagy-related 7 (Atg7) and microtubule-associated protein 1A/1B-light chain 3II (LC3II). MA also increased the phosphorylation levels of Wnt/β-catenin proteins, such as GSK3β (Ser9) and β-catenin (Ser552 and Ser675). Treatment with XAV939, an inhibitor of the Wnt/β-catenin pathway, attenuated the MA-induced increase in β-catenin nuclear translocation. Moreover, XAV939 reduced MA-induced effects on cell cycle progression, autophagy, and DPC proliferation. On the other hand, MA increased the levels of phospho (Thr202/Tyr204)-extracellular signal regulated kinases (ERK). MA-induced ERK phosphorylation led to changes in the expression levels of Cdc2, Atg7 and LC3II, as well as DPC proliferation. Our results suggest that MA promotes anagen signaling via autophagy and cell cycle progression by activating the Wnt/β-catenin and ERK pathways in DPCs.

• Journal of Ginseng Research
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• 제46권6호
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• pp.738-749
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• 2022

Background: Ginseng possesses antitumor effects, and ginsenosides are considered to be one of its main active chemical components. Ginsenosides can further be hydrolyzed to generate secondary saponins, and 20(R)-panaxotriol is an important sapogenin of ginsenosides. We aimed to synthesize a new ginsengenin derivative from 20(R)-panaxotriol and investigate its antitumor activity in vivo and in vitro. Methods: Here, 20(R)-panaxotriol was selected as a precursor and was modified into its derivatives. The new products were characterized by ¹H-NMR, ¹³C-NMR and HR-MS and evaluated by molecular docking, MTT, luciferase reporter assay, western blotting, immunofluorescent staining, colony formation assay, EdU labeling and immunofluorescence, apoptosis assay, cells migration assay, transwell assay and in vivo antitumor activity assay. Results: The derivative with the best antitumor activity was identified as 6,12-dihydroxy-4,4,8,10,14-pentamethyl-17-(2,6,6-trimethyltetrahydro-2H-pyran-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl(tert-butoxycarbonyl)glycinate (A11). The focus of this research was on the antitumor activity of the derivatives. The efficacy of the derivative A11 (IC₅₀ < 0.3 µM) was more than 100 times higher than that of 20(R)- panaxotriol (IC₅₀ > 30 µM). In addition, A11 inhibited the protein expression and nuclear accumulation of the hypoxia-inducible factor HIF-1α in HeLa cells under hypoxic conditions in a dose-dependent manner. Moreover, A11 dose-dependently inhibited the proliferation, migration, and invasion of HeLa cells, while promoting their apoptosis. Notably, the inhibition by A11 was more significant than that by 20(R)-panaxotriol (p < 0.01) in vivo. Conclusion: To our knowledge, this is the first study to report the production of derivative A11 from 20(R)-panaxotriol and its superior antitumor activity compared to its precursor. Moreover, derivative A11 can be used to further study and develop novel antitumor drugs.