• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.329-334
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• 2006

Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. In this report, we describe a new synthesized cholesterolic cationic lipid (2-aminoethylcarbamate-cholesterol) and dioleylphosphatidylethanolamine (DOPE) improve the cellular uptake properties of antisense ODNs, as well as plasmid DNA with low toxicity. This formulation of cholesterolic cationic lipid termed Chol-E, efficiently transfects ODNs and plasmids into many cell types in the presense or absence of 10% serum in the medium.

• Nutrition Research and Practice
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• v.3 no.3
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• pp.192-199
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• 2009

Cadmium intoxication has been associated with the dysregulation of iron homeostasis. In the present study, we investigated the effect of cadmium on the expression of ferroportin 1 (FPN1), an important iron transporter protein that is involved in iron release from macrophages. When we incubated cadmium with J774 mouse macrophage cells, FPN1 mRNA levels were significantly increased in a dose- and time-dependent manner. Furthermore, the cadmium-induced FPN1 mRNA expression was associated with increased levels of FPN1 protein. On the other hand, cadmium-mediated FPN1 mRNA induction in J774 cells was completely blocked when cells were co-treated with a transcription inhibitor, acitomycin D. Also, cadmium directly stimulated the activity of the FPN1-promoter driven luciferase reporter, suggesting that the cadmium up-regulates FPN1 gene expression in a transcription-dependent manner. Finally, cadmium exposure to J774 macrophages increased intracellular reactive oxygen species (ROS) levels by ${\sim}2$-fold, compared to untreated controls. When J774 cells were co-treated with antioxidant N-acetylcystein, the cadmium-induced FPN1 mRNA induction was significantly attenuated. In summary, the results of this study clearly demonstrated that cadmium increased FPN1 expression in macrophages through a mechanism that involves ROS production, and suggests another important interaction between iron and cadmium metabolism.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.48-54
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• 2005

A comparison of promoter fusions with the luxCDABE genes from Vibrio fischeri and Photorhabdus luminescens was made using promoters from several genes (katG, sodA, and pqi-5) of E. coli that are responsive to oxidative damage. The respective characteristics, such as the basal and maximum bioluminescence and the relative bioluminescence, were compared. E. coli strains carrying fusions of the promoters to P. luminescens lux showed higher basal and maximally induced bioluminescent levels than strains carrying the same promoter fused to the luxCDABE genes from V. fischeri. The sensitivities between the strains were similar, regardless of the luciferase used, but lower response ratios were seen from strains harboring the P. luminescens lux fusions. Furthermore, using the two katG::lux fusion strains, the bioluminescence from the P. luminescens lux fusion strain, DK1, was stable after reaching a maximum, while that of strain DPD2511 decreased very rapidly due to substrate limitation.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.155-162
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• 2004

In mammalian, cytochrome P4501A1 (CYP1A1) is very important for metabolism of xenobiotics such as PAHs(Polycyclic aromatic hydrocarbon) and heterocyclic amine, and it is induced by environmental contaminants such as PAHs, TCDD(2,3,7,8-tetrchlorodibenzo-p-dioxin) and 3-MC (3-methylcholanthrene). In fish, like mammalian, when it is exposed to environmental contaminants, they cause specific and sensitive induction of CYP1A. Therefore, induction of CYP1A in fish and mammalian is widely used as a biomarker for exposure of environmental contaminants. In this study, to compare the function of Cyp1a1 in fish with it in mammalian, we have used rainbow trout(Oncorhynchys mykiss) hepatoma cells (RTH-149) and mouse hepatocyte (Hepa-I). in order to examine induction of Cyp1a1 by TCDD, we have used the bioassay system. We examined effects of TCDD on the Cyp1a1-luciferase reporter gene activity, 7-ethoxyresorufin O-deethylase(EROD) activity and Cypa mRNA level.

• Molecules and Cells
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• v.37 no.5
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• pp.389-398
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• 2014

Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between siah and Drosophila Siah homologue (sina) suggests their important physiological roles during embryonic development. However, detailed functional studies of Siah in vertebrate development have not been carried out. Here we report that Siah2 specifically augments nodal related gene expression in marginal blastomeres at late blastula through early gastrula stages of zebrafish embryos. Siah2 dependent Nodal signaling augmentation is confirmed by cell-based reporter gene assays using 293T cells and 3TP-luciferase reporter plasmid. We also established a molecular hierarchy of Siah as a upstream regulator of FoxH1/Fast1 transcriptional factor in Nodal signaling. Elevated expression of nodal related genes by overexpression of Siah2 was enough to override the inhibitory effects of atv and lft2 on the Nodal signaling. In particular, E3 ubiquitin ligase activity of Siah2 is critical to limit the duration and/or magnitude of Nodal signaling. Additionally, since the embryos injected with Siah morpholinos mimicked the atv overexpression phenotype at least in part, our data support a model in which Siah is involved in mesendoderm patterning via modulating Nodal signaling.

• Nutrition Research and Practice
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• v.2 no.4
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• pp.317-321
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• 2008

Macrophages play a key role in iron metabolism by recycling iron through erythrophagocytosis. Ferroportin-l (FPN1) is a transporter protein that is known to mediate iron export from macrophages. Since divalent metals often interact with iron metabolism, we examined if divalent metals could regulate the expression of FPN1 in macrophages. J774 macrophage cells were treated with copper, manganese, zinc, or cobalt at 10, 50, or $100\;{\mu}M$ for 16 to 24 h. Then, FPN1 mRNA and protein levels were determined by quantitative real-time PCR and Western blot analyses, respectively. In addition, effects of divalent metals on FPN1 promoter activity were examined by luciferase reporter assays. Results showed that copper significantly increased FPN1 mRNA levels in a dose-dependent manner. The copper-induced expression of FPN1 mRNA was associated with a corresponding increase in FPN1 protein levels. Also, copper directly stimulated the activity of FPN1 promoter-driven reporter construct. In contrast, manganese and zinc had no effect on the FPN1 gene expression in J774 cells. Interestingly, cobalt treatment in J774 cells decreased FPN1 protein levels without affecting FPN1 mRNA levels. In conclusion, our study results demonstrate that divalent metals differentially regulate FPN1 expression in macrophages and indicate a potential interaction of divalent metals with the FPN1-mediated iron export in macrophages.

• Journal of Microbiology and Biotechnology
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• v.29 no.7
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• pp.1137-1143
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• 2019

Hepatitis E virus (HEV) accounts for 20 million infections in humans worldwide. In most cases, the infections are self-limiting while HEV genotype 1 infection cases may lead to lethal infections in pregnant women (~ 20% fatality). The lack of small animal models has hampered detailed analysis of virus-host interactions and HEV-induced pathology. Here, by employing a recently developed culture-adapted HEV, we demonstrated that methyltransferase, a non-structural protein, strongly inhibits melanoma differentiation-associated gene 5 (MDA5)-mediated activation of type I interferon responses. Compared to uninfected controls, HEV-infected cells display significantly lower levels of $IFN-{\beta}$ promoter activation when assessed by luciferase assay and RT-PCR. HEV genome-wide screening showed that HEV-encoded methyltransferase (MeT) strongly inhibits MDA5-mediated transcriptional activation of $IFN-{\beta}$ and $NF-{\kappa}B$ in a dose-responsive manner whether or not it is expressed in the presence/absence of a tag fused to it. Taken together, current studies clearly demonstrated that HEV MeT is a novel antagonist of MDA5-mediated induction of $IFN-{\beta}$ signaling.

• Molecules and Cells
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• v.27 no.1
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• pp.113-118
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• 2009

In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5'-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-${\kappa}B$, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-${\kappa}B$ binding site at -731 to -722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and $G{\ddot{O}}6976$, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.207-212
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• 2012

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

• Biomedical Science Letters
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• v.12 no.3
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• pp.131-137
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• 2006

RGS is a negative regulator of G-protein signaling and can be identified by the presence of a conserved $120{sim}125$ amino acid motif, which is referred to as the RGS box. A number of RGSs are induced in response to a wide variety of stimuli. Increased levels of RGSs lead to significant decreases in GPCR responsiveness. To obtain further evidence of a role of RGS proteins in rat C6 astrocytoma cells, we first determined the expression profile of RGS-specific mRNA in C6 cells using reverse transcription-polymerase chain reaction (RT-PCR) with a poly dT18 primer and transcript-specific primers. We found that RGS2, RGS3, RGS6, RGS9, RGS10, RGS12, and RGS16 were differentially expressed in C6 astrocytoma cells. The highest expression rate was found for RGS3, followed by RGS16, RGS10 and RGS9, whereas the expression level for RGS2 was barely detectable. We next assessed whether forskolin regulated the expression of RGSs expressed in C6 astrocytoma cells. The present study found that forskolin dose-dependently stimulated the expression of RGS2 transcripts. This up-regulation of RGS2 gene was abrogated by H-89, potent and broad-spectrum protein kinase A (PKA) inhibitors. Actinomycin D completely inhibited the up-regulation of RGS2 gene induced by forskolin $(10{\mu}M)$, indicating that the regulation of RGS2 gene is controlled at the transcriptional level. In addition, forskolin did significantly activate transcriptional cAMP response element (CRE) in either HEK 293 cells or C6 cells and did not modulate the $NF-{\kappa}B$ and AP-l activity as measured by luciferase reporter gene assay. Finally, forskolin induced the expression of RGS2 mRNA in C6 astrocytoma cells, which depend on the PKA pathway and CRE transcriptional pathways.