• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1452-1459
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• 2007

The Dps protein, which is overexpressed in harsh environments, is known to playa critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region of the S. typhimurium dps gene. The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. The fur mutant, $_X4659$, evidenced a reduced level of ${\beta}$-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (${\Delta}dps$) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.

• Molecules and Cells
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• v.21 no.2
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• pp.261-268
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• 2006

The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 ~ -272 (PE1) and +28 ~ +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth-luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein.

• Journal of Ginseng Research
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• v.46 no.2
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• pp.248-254
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• 2022

Background: Zinc homeostasis is essential for human health and is regulated by several zinc transporters including ZIP and ZnT. ZIP4 is expressed in the small intestine and is important for zinc absorption from the diet. We investigated in the present study the effects of Panax ginseng (P. ginseng) extract on modulating Zip4 expression and cellular zinc levels in mouse Hepa cells. Methods: Hepa cells were transfected with a luciferase reporter plasmid that contains metal-responsive elements, incubated with P. ginseng extract, and luciferase activity was measured. Using ⁶⁵ZnCl₂, zinc uptake in P. ginseng-treated cells was measured. The expression of Zip4 mRNA and protein in Hepa cells was also investigated. Finally, using a luciferase reporter assay system, the effects of several ginsenosides were monitored. Results: The luciferase activity in cells incubated with P. ginseng extract was significantly higher than that of control cells cultured in normal medium. Hepa cells treated with P. ginseng extract exhibited higher zinc uptake. P. ginseng extract induced Zip4 mRNA expression, which resulted in an enhancement of Zip4 protein expression. Furthermore, some ginsenosides, such as ginsenoside Rc and Re, enhanced luciferase activity driven by intracellular zinc levels. Conclusion: P. ginseng extract induced Zip4 expression at the mRNA and protein level and resulted in higher zinc uptake in Hepa cells. Some ginsenosides facilitated zinc influx. On the basis of these results, we suggest a novel effect of P. ginseng on Zip4-mediated zinc influx, which may provide a new strategy for preventing zinc deficiency.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.3
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• pp.226-233
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• 2007

Purpose: Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. Materials and Methods: A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. Results: The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, $T_{1/2}$ of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. Conclusion: A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.

• Journal of Ginseng Research
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• v.21 no.3
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• pp.141-146
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• 1997

The role of cAMP in the differentiation process of F9 cells induced by ginsenosides was examined by performing transient transfixion assay with CRE-luciferase reporter plasmid, GR thansactivation assay with GRE-luciferase activity with or without treatment of CAMP and forskolin, an activator of adenylate cyclase, and protein klnase A assay in the presence of ginsenosides. Ginsenosides had no effect on CRE-transactivation activity, whereas retinoic acid induced the activity. When cAMP or forskolin was treated with ginsenosides, GRE-luciferase activity was further augumented by them. In addition, ginsenosides induced protein kinase A activity in the presence of cAMP. These results suggest that ginsenosides activate cAMP-dependent protein kinase A which, in turn, increase GR activity in F9 cells.

• BMB Reports
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• v.42 no.3
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• pp.154-159
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• 2009

JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.356-363
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• 2015

In order to study genetic engineering in trees, the characterization of genes and promoters from trees is necessary. We isolated the promoter region (867 bp) of Pagns-LTP from poplar (P. alba ${\times}$ P. glandulosa) and characterized its activity in transgenic poplar plants using a ${\beta}$-glucuronidase (GUS) reporter gene. High-level expression of the Pagns-LTP transcript was found in poplar roots, while comparatively low-level expression was found in the young leaves. Pagns-LTP mRNA was not detected in other poplar tissues. Additionally, transgenic poplar plants that contained a Pagns-LTP promoter fused to a GUS reporter gene, displayed tissue-specific GUS enzyme activity localized in root tissue. In silico analysis of the Pagns-LTP promoter sequence reveals the presence of several cis-regulatory elements responsive to phytohormones, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrate that the Pagns-LTP promoter has tissue-specific expression activity in poplar roots and leaves that may be involved in organ development and plant resistance to various stresses. Therefore, we anticipate that the Pagns-LTP promoter would be a useful tool to genetically optimize woody plants for functional genomics.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.503-510
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• 2015

The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a light-oxygen-voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu²⁺ was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 µM. In addition, Cu²⁺-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu²⁺ binding with the iLOV protein.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1646-1649
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• 2006

Protein synthesis is the ultimate outcome of gene expression which, in turn, is regulated by several translation factors. We attempted to identify substances that can inhibit the translation process in vitro when the outcome protein is luciferase. To this end, we developed a sensitive cell-free protein synthesis assay using luciferase as the reporter. The synthesis of luciferase increased proportionately as mRNA was added to a $15-{\mu}l$reaction medium in concentrations raging from 5 ng to 500 ng. The maximum amount of luciferase was synthesized when the media were incubated at $25^{\circ}C$ for 40 min. The concentration of each compound that inhibited luciferase production by 50% ($IC_{50}$) was calculated. Hygromycin, puromycin, and cycloheximide yielded an $IC_{50}$ of 0.008, 0.8, and $0.7{\mu}g/ml$, respectively. A filtrate of Streptomyces spp. isolates inhibited protein synthesis up to S-fold when added to the in vitro translation assay mixture.