• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.449-456
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• 2017

Beauvericin (BEA), a cyclic hexadepsipeptide produced by the fungus Beauveria bassiana, is known to have anti-cancer, anti-inflammatory, and anti-microbial actions. However, how BEA suppresses macrophage-induced inflammatory responses has not been fully elucidated. In this study, we explored the anti-inflammatory properties of BEA and the underlying molecular mechanisms using lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells. Levels of nitric oxide (NO), mRNA levels of transcription factors and the inflammatory genes inducible NO synthase (iNOS) and interleukin (IL)-1, and protein levels of activated intracellular signaling molecules were determined by Griess assay, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), luciferase reporter gene assay, and immunoblotting analysis. BEA dose-dependently blocked the production of NO in LPS-treated RAW264.7 cells without inducing cell cytotoxicity. BEA also prevented LPS-triggered morphological changes. This compound significantly inhibited nuclear translocation of the $NF-{\kappa}B$ subunits p65 and p50. Luciferase reporter gene assays demonstrated that BEA suppresses MyD88-dependent NF-${\kappa}B$ activation. By analyzing upstream signaling events for $NF-{\kappa}B$ activation and overexpressing Src and Syk, these two enzymes were revealed to be targets of BEA. Together, these results suggest that BEA suppresses $NF-{\kappa}B$-dependent inflammatory responses by suppressing both Src and Syk.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.174-178
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• 2011

In Escherichia coli, DcuS/R two-component system controls fumarate import and utilization related gene expression. To understand the dynamic response of the bacterium DcuS/R two-component system with respect to fumarate concentrations, DcuS/R induced dctA promoter was integrated with GFP reporter protein. Expression monitoring study using recombinant strain showed that dctA promoter was upregulated with 1 mM of fumarate in M9 minimal medium.

• Toxicological Research
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• v.23 no.2
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• pp.123-126
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• 2007

We isolated a lignan, kobusin from Geranium thunbergii and studied its effect on the expression of inducible nitric oxide synthase (iNOS) gene in a monocyte/macrophage cell line, RAW264.7 cells. Kobusin inhibited lipopolysaccharide (LPS)-stimulated NO production and the expression of iNOS in a concentration-dependent manner. To identify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of kobusin on both the luciferase reporter activity using $NF-{\kappa}B$ minimal promoter and the nuclear translocation of p65. Kobusin suppressed the reporter gene activity and the LPS-induced movement of p65 in to nucleus. $NF-{\kappa}B$ activation is controlled by the phosphorylation and subsequent degradation of $I-{\kappa}B{\alpha}$, and in the present study, we found that $I-{\kappa}B{\alpha}$ phosphorylation was also inhibited by kobusin. Our findings indicate that kobusin may provide a developmental basis for an agent against inflammatory diseases.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.568-574
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• 1991

The cloned glyceraldehyde-3-phosphate dehydrogenase (GAP) gene of Saccharomyces cereviszae (Holland et al., 1983) has been characterized. Based on the communication, we have also cloned 2.1 kb CAP DNA fragment and modified this fragment as a portable promoter. Two yeast expression vectors, one is YCp type vector being maintained at low copy number (1 or 2) and the other is YEp type vector at high copy number, have been constructed with the GAP promoter and the PH05' gene as a reporter. Our plasrnids were introduc,ed into S. cerevisiae HY-1, which has been improved. The $Trp^+$ transformants expressed APase activity efficiently and showed high level of PH05' transcripts.

• Nutrition Research and Practice
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• v.14 no.5
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• pp.453-462
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• 2020

BACKGROUND/OBJECTIVES: Platycodon grandiflorum (PG), an oriental herbal medicine, has been known to improve liver function, and has both anti-inflammatory and antimicrobial properties. However, little is known about the immune-enhancing effects of PG and its mechanism. In this study, we aimed to investigate whether fermented PG extract (FPGE), which has increased platycodin D content, activates the immune response in a murine macrophage cell line, RAW 264.7. MATERIALS/METHODS: Cell viability was determined by Cell Counting Kit-8 assay and the nitric oxide (NO) levels were measured using Griess reagent. Cytokine messenger RNA levels of were monitored by quantitative reverse transcription polymerase chain reaction. To investigate the molecular mechanisms underlying immunomodulatory actions of FPGE in RAW 264.7 cells, we have conducted luciferase reporter gene assay and western blotting. RESULTS: We found that FPGE treatment induced macrophage cell proliferation in a dose-dependent manner. FPGE also modulated the expression of NO and pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The activation and phosphorylation levels of nuclear factor kappa B (NF-κB) were increased by FPGE treatment. Moreover, 5-aminoimidazole-4-carboxamide ribonucleotide, an activator of AMP-activated kinase (AMPK), significantly reduced both lipopolysaccharides- and FPGE-induced NF-κB reporter gene activity. CONCLUSIONS: Taken together, our findings suggest that FPGE may be a novel immune-enhancing agent acting via AMPK-NF-κB signaling pathway.

• Journal of Aquaculture
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• v.13 no.1
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• pp.29-37
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• 2000

In previously study we isolated several fish matrix attachment regions (MARs) capable of replicating the plasmid by itself. In this study we construct a fish expression vector pBaEGFP(+) containing mud loach ${\beta}$-actin promoter EGFP as reporter gene and SV40 signal. To analyze the effects of the fish expression vector respectively. The fish ARS containing constructs pBaEGFP(+)-ARSs were transfected cells with pBaEGFP(+)-ARS101 and pBaEGFP(+)-ARS223 reduced 10 days to 25 days and then was constant to 30 days after transfection while that of the control vector without ARS element was basal level. The intensity of both constructs showed about 30fold of the intensity compared with the control vector on 30days after transfection individually .E. coli back-transformation analysis shows that pBaEGFP(+)-ARS223 and pBaEGFP(+)-ARS905 maintain in episomal state at least 30 days after transfection. The result indicates that both may be able to replicate the vector in BF-2 cell. Therefore the matrix-attached ARSs enhancing expression of the reporter gene might be useful as a component o the expression vector for transgenic studies.

• 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.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.201-205
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• 1995

The synthetic oligomers called nos right palindrome (RP) element and left palindrome (LP) element were inserted into nos.minimal promoter nos 5'-101 deletion mutant The activity of nos promoter was measured by studying the expression pattern of gene fusion between nos promoter and reporter genes such as chloramphenicol acetyltransferase and $\beta$-glucuconidase. Analysis of transgenic tobacco plane carrying transgene showed that the activity of nos minimal promoter activity was recovered by insertion of synthetic nos RP element. Nos RP element insertion of nos minimal promoter was induced by auxin, dithiothreitol, salicylic acid and methyl jasmonate.

• 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.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.18-24
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• 2001

Expression of the baculovirus major envelope glycoprotein gene(gp64) is regulated by transcription from botha early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1(BmNPV-K1) was characterized. The gp64 gene was local-ized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcorR 1-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E.coli lacZ gene was introduced onto pBm64 as a reporter gene and expressed transiently in B. mori 5(Bm 5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of $\beta$-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of $\beta$-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.