• Title/Summary/Keyword: Reporter gene assay

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Expression characterization and transcription regulation analysis of porcine Yip1 domain family member 3 gene

  • Ni, Dongjiao;Huang, Xiang;Wang, Zhibo;Deng, Lin;Zeng, Li;Zhang, Yiwei;Lu, Dongdong;Zou, Xinhua
    • Asian-Australasian Journal of Animal Sciences
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    • v.33 no.3
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    • pp.398-407
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    • 2020
  • Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.

Wnt7b is Upregulated in Macrophages during Thymic Regeneration and Negatively Regulated by RANKL (흰쥐 가슴샘 재생과정 동안 대식세포에서 Wnt 7b의 발현증가 및 RANKL에 의한 발현조절)

  • Kim, Jong-Gab;Kim, Sung-Min;Kim, Bong-Seon;Kim, Jae-Bong;Yoon, Sik;Bae, Soo-Kyung
    • Journal of Life Science
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    • v.17 no.7 s.87
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    • pp.923-930
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    • 2007
  • Thymus can regenerate to its normal mass within 14 days after acute involution induced by cyclophosphamide (CY) in adult rat. Despite the established role of Wnt pathways in the process of thymus development, they have not yet been associated with the regeneration of adult thymus. The purpose of this study was to investigate whether Wnt7b, which is expressed in developing thymic epithelial cells rather than in thymocytes, is modulated during thymic regeneration in adult rat. Here, we show that Wnt7b expression was up-regulated in the regenerating thymus. Cells immunolabeled for the Wnt7b were identified as macrophages. Furthermore, Wnt7b gene expression was decreased by the treatment of receptor activator of NF-kappaB ligand (RANKL). Taken together, our results demonstrate that Wnt7b gene expression was increased in macrophages during thymic regeneration and negatively regulated by RANKL.

Functional Analysis of the Stress-Inducible Soybean Calmodulin Isoform-4 (GmCaM-4) Promoter in Transgenic Tobacco Plants

  • Park, Hyeong Cheol;Kim, Man Lyang;Kang, Yun Hwan;Jeong, Jae Cheol;Cheong, Mi Sun;Choi, Wonkyun;Lee, Sang Yeol;Cho, Moo Je;Kim, Min Chul;Chung, Woo Sik;Yun, Dae-Jin
    • Molecules and Cells
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    • v.27 no.4
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    • pp.475-480
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    • 2009
  • The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

MicroRNA analysis reveals the role of miR-214 in duck adipocyte differentiation

  • Wang, Laidi;Hu, Xiaodan;Wang, Shasha;Yuan, Chunyou;Wang, Zhixiu;Chang, Guobin;Chen, Guohong
    • Animal Bioscience
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    • v.35 no.9
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    • pp.1327-1339
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    • 2022
  • Objective: Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation. Methods: Successful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription. Results: There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3'untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2. Conclusion: Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

MicroRNA-23b is a Potential Tumor Suppressor in Diffuse Large B-cell Lymphoma (미만성 거대 B 세포 림프종(DLBCL)에서 microRNA-23b의 잠재적 종양 억제자로서의 효과)

  • Nam, Jehyun;Kim, Eunkyung;Kim, Jinyoung;Jeong, Dawoom;Kim, Donguk;Kwak, Bomi;Kim, Sang-Woo
    • Journal of Life Science
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    • v.27 no.2
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    • pp.149-154
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    • 2017
  • Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-hodgkin lymphoma. Advances in the chemotherapeutic treatment of this disease have improved the outcomes of DLBCL; nonetheless, many patients still die of DLBCL, and therefore, a better understanding of this disease and identification of novel therapeutic targets are urgently required. In a recent gene expression profiling study, PDE (phosphodiesterase) 4B was found to be overexpressed in chemotherapy-resistant tumors. The major function of PDE4B is to inactivate the second messenger cyclic 3',5' monophosphate (cAMP) by catalyzing the hydrolysis of cAMP to 5'AMP. It is known that cAMP induces cell cycle arrest and/or apoptosis in B cells, and PDE4B abolishes cAMP's effect on B cells. However, the mechanism by which PDE4B is overexpressed remains unclear. Here, we show that the aberrant expression of miRNA may be associated with the overexpression of this gene. The PDE4B 3' untranslated region (UTR) has three functional binding sites of miR-23b, as confirmed by luciferase reporter assays. Interestingly, miR-23b-binding sites were evolutionarily conserved from humans to lizards, implying the critical role of PDE4B-miR-23b interaction in cellular physiology. The ectopic expression of miR-2 3b repressed PDE4B mRNA levels and enhanced intracellular cAMP concentrations. Additionally, miR-23b expression inhibited cell proliferation and survival of DLBCL cells only in the presence of forskolin, an activator of adenylyl cyclase, suggesting that miR-23b's effect is via the downregulation of PDE4B. These results together suggest that miR-23b could be a therapeutic target for overcoming drug resistance by repressing PDE4B in DLBCL.

An International Collaborative Program To Discover New Drugs from Tropical Biodiversity of Vietnam and Laos

  • Soejarto, Djaja D.;Pezzuto, John M.;Fong, Harry H.S.;Tan, Ghee Teng;Zhang, Hong Jie;Tamez, Pamela;Aydogmus, Zeynep;Chien, Nguyen Quyet;Franzblau, Scott G.;Gyllenhaal, Charlotte;Regalado, Jacinto C.;Hung, Nguyen Van;Hoang, Vu Dinh;Hiep, Nguyen Tien;Xuan, Le Thi;Hai, Nong Van;Cuong, Nguyen Manh;Bich, Truong Quang;Loc, Phan Ke;Vu, Bui Minh;Southavong, Boun Hoong
    • Natural Product Sciences
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    • v.8 no.1
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    • pp.1-15
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    • 2002
  • An International Cooperative Biodiversity Group (ICBG) program based at the University of Illinois at Chicago initiated its activities in 1998, with the following specific objectives: (a) inventory and conservation of of plants of Cuc Phuong National Park in Vietnam and of medicinal plants of Laos; (b) drug discovery (and development) based on plants of Vietnam and Laos; and (c) economic development of communities participating in the ICBG project both in Vietnam and Laos. Member-institutions and an industrial partner of this ICBG are bound by a Memorandum of Agreement that recognizes property and intellectual property rights, prior informed consent for access to genetic resources and to indigenous knowledge, the sharing of benefits that may arise from the drug discovery effort, and the provision of short-term and long-term benefits to host country institutions and communities. The drug discovery effort is targeted to the search for agents for therapies against malaria (antimalarial assay of plant extracts, using Plasmodium falciparum clones), AIDS (anti-HIV-l activity using HOG.R5 reporter cell line (through transactivation of the green fluorescent protein/GFP gene), cancer (screening of plant extracts in 6 human tumor cell lines - KB, Col-2, LU-l, LNCaP, HUVEC, hTert-RPEl), tuberculosis (screening of extracts in the microplate Alamar Blue assay against Mycobacterium tuberculosis $H_{37}Ra\;and\;H_{37}Rv),$ all performed at UIC, and CNS-related diseases (with special focus on Alzheimer's disease, pain and rheumatoid arthritis, and asthma), peformed at Glaxo Smith Kline (UK). Source plants were selected based on two approaches: biodiversity-based (plants of Cuc Phuong National Park) and ethnobotany-based (medicinal plants of Cuc Phuong National Park in Vietnam and medicinal plants of Laos). At mc, as of July, 2001, active leads had been identified in the anti-HIV, anticancer, antimalarial, and anti- TB assay, after the screening of more than 800 extracts. At least 25 biologically active compounds have been isolated, 13 of which are new with anti-HIV activity, and 3 also new with antimalarial activity. At GSK of 21 plant samples with a history of use to treat CNS-related diseases tested to date, a number showed activity against one or more of the CNS assay targets used, but no new compounds have been isolated. The results of the drug discovery effort to date indicate that tropical plant diversity of Vietnam and Laos unquestionably harbors biologically active chemical entities, which, through further research, may eventually yield candidates for drug development. Although the substantial monetary benefit of the drug discovery process (royalties) is a long way off, the UIC ICBG program provides direct and real-term benefits to host country institutions and communities.

Identification of the Plant Part of Gleditsia sinensis that Activates Nrf2, an Anti-oxidative Transcription Factor (조협의 부위에 따른 항산화 전사인자 Nrf2 활성 효과)

  • Choi, Jiyeon;Kim, Kyun Ha;Choi, Jun Yong;Han, Chang Woo;Ha, Ki Tae;Jeong, Han-Sol;Joo, Myungsoo
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.28 no.3
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    • pp.303-309
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    • 2014
  • The fruit of Gleditsia sinensis has been extensively used as a key ingredient of an herbal remedy for the treatment of various inflammatory diseases in traditional Korean Medicine. However, the reason of using the fruit of G. sinensis for the remedy is unclear. Since Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key anti-inflammatory transcription factor, which is activated by the fruit of G. sinesis, we examined whether other plant parts of G. sinensis are also capable of suppressing inflammatory responses by activating Nrf2. Water extracts of various parts of G. sinensis were prepared and tested for Nrf2 activation by reporter assay and western blot analysis. Our results show that the hull of G. sinensis is the most potent in activating Nrf2. Sequential organic solvent extraction of the hull show that all the fractions had a higher potency in activating Nrf2 than the water extract, albeit differential degrees. The hull originated from Korea in general activated Nrf2 strongly compared to that of China. Chloroform fraction of the hull was further examined, showing that the fraction induced nuclear localization of Nrf2, indicative of activated Nrf2, and Nrf2-dependent gene expression including NAD(P)H dehydrogenase quinone 1 (NQO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and heme oxygenase - 1 (HO-1). Therefore, our results show that, among other plant parts examined in this study, the hull of G. sinensis is the most potent, providing the experimental basis for the use of the hull of G. sinensis as an active ingredient for an anti-inflammatory remedy.

Pyrrole-Derivative of Chalcone, (E)-3-Phenyl-1-(2-Pyrrolyl)-2-Propenone, Inhibits Inflammatory Responses via Inhibition of Src, Syk, and TAK1 Kinase Activities

  • Yang, Sungjae;Kim, Yong;Jeong, Deok;Kim, Jun Ho;Kim, Sunggyu;Son, Young-Jin;Yoo, Byong Chul;Jeong, Eun Jeong;Kim, Tae Woong;Han Lee, In-Sook;Cho, Jae Youl
    • Biomolecules & Therapeutics
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    • v.24 no.6
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    • pp.595-603
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    • 2016
  • (E)-3-Phenyl-1-(2-pyrrolyl)-2-propenone (PPP) is a pyrrole derivative of chalcone, in which the B-ring of chalcone linked to ${\beta}$-carbon is replaced by pyrrole group. While pyrrole has been studied for possible Src inhibition activity, chalcone, especially the substituents on the B-ring, has shown pharmaceutical, anti-inflammatory, and anti-oxidant properties via inhibition of NF-${\kappa}B$ activity. Our study is aimed to investigate whether this novel synthetic compound retains or enhances the pharmaceutically beneficial activities from the both structures. For this purpose, inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 cells were analyzed. Nitric oxide (NO) production, inducible NO synthase (iNOS) and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) mRNA expression, and the intracellular inflammatory signaling cascade were measured. Interestingly, PPP strongly inhibited NO release in a dose-dependent manner. To further investigate this anti-inflammatory activity, we identified molecular pathways by immunoblot analyses of nuclear fractions and whole cell lysates prepared from LPS-stimulated RAW264.7 cells with or without PPP pretreatment. The nuclear levels of p50, c-Jun, and c-Fos were significantly inhibited when cells were exposed to PPP. Moreover, according to the luciferase reporter gene assay after cotransfection with either TRIF or MyD88 in HEK293 cells, NF-${\kappa}B$-mediated luciferase activity dose-dependently diminished. Additionally, it was confirmed that PPP dampens the upstream signaling cascade of NF-${\kappa}B$ and AP-1 activation. Thus, PPP inhibited Syk, Src, and TAK1 activities induced by LPS or induced by overexpression of these genes. Therefore, our results suggest that PPP displays anti-inflammatory activity via inhibition of Syk, Src, and TAK1 activity, which may be developed as a novel anti-inflammatory drug.

The Effect of Magnolol on UVB-induced Inflammation Damage Control via the Nrf2-SOCS3-Jak2-STAT3 Pathway in Human Dermal Fibroblasts (마그놀롤의 HDF세포에서 Nrf2-SOCS3-Jak2-STAT3에 의한 UVB 유래 염증데미지 조절)

  • Nam, Young sun;Ji, Juree
    • Journal of Life Science
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    • v.30 no.10
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    • pp.867-876
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    • 2020
  • This study investigated the repair of UVB-induced cell damage by magnolol. We performed a drug-repurposing screen, and, in the STAT3 reporter gene assay, magnolol was identified as a suppressor of STAT3 that improves the cell viability of HDF cells. HDF cells treated with IL-6, UVB, and IFNγ showed the highest expression of Jak2 and phosphorylated STAT3 (p-STAT3), and magnolol was able to decrease the expression of Jak2 and p-STAT3 in UVB-induced cells. Moreover, UVB-damaged cell growth increased significantly in correlation with both reactivation and with magnolol in a dose-dependent manner. Compared with AG490 (a Jak2 inhibitor) treatment of UVB-treated HDF cells, cell proliferation increased significantly. We confirmed that AG490 and magnolol reduced TNF-α concentrations, and Western blotting (protein level) showed decreases in Jak2 and p-STAT3 expression in only the magnolol-treated cells. The expression of Jak2, p-STAT3, and SOCS3 also increased only after treatment with magnolol. Cells were treated with magnolol and ML385 (an NRF2 inhibitor), and these secondary metabolites reduced cell proliferation and NRF2 expression. The amount of MMP9 was also increased by cotreatment with magnolol and ML385. Collectively, these results demonstrate the potential of magnolol for repairing cells after UVB-induced damage by regulating the expression of NRF2, SOCS3, Jak2, and STAT3.

Induction of Nrf2/ARE-mediated cytoprotective genes by red ginseng oil through ASK1-MKK4/7-JNK and p38 MAPK signaling pathways in HepG2 cells

  • Bak, Min Ji;Truong, Van-Long;Ko, Se-Yeon;Nguyen, Xuan Ngan Giang;Jun, Mira;Hong, Soon-Gi;Lee, Jong-Won;Jeong, Woo-Sik
    • Journal of Ginseng Research
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    • v.40 no.4
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    • pp.423-430
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    • 2016
  • Background: The induction of cellular defensive genes such as phase II detoxifying and antioxidant enzymes is a highly effective strategy for protection against carcinogenesis as well as slowing cancer development. Transcription factor Nrf2 (nuclear factor E2-related factor 2) is responsible for activation of phase II enzymes induced by natural chemopreventive compounds. Methods: Red ginseng oil (RGO) was extracted using a supercritical $CO_2$ extraction system and chemical profile of RGO was investigated by GC/MS. Effects of RGO on regulation of the Nrf2/antioxidant response element (ARE) pathway were determined by ARE-luciferase assay, western blotting, and confocal microscopy. Results: The predominant components of RGO were 9,12-octadecadienoic acid (31.48%), bicyclo[10.1.0] tridec-1-ene (22.54%), and 22,23-dihydrostigmasterol (16.90%). RGO treatment significantly increased nuclear translocation of Nrf2 as well as ARE reporter gene activity, leading to upregulation of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Phosphorylation of the upstream kinases such as apoptosis signal-regulating kinase (ASK)1, mitogen-activated protein kinase (MAPK) kinase (MKK)4/7, c-Jun N-terminal kinase (JNK), and p38 MAPK were enhanced by treatment with RGO. In addition, RGO-mediated Nrf2 expression and nuclear translocation was attenuated by JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190. Conclusion: RGO could be used as a potential chemopreventive agent, possibly by induction of Nrf2/ARE-mediated phase II enzymes via ASK1-MKK4/7-JNK and p38 MAPK signaling pathways.