• Journal of Microbiology
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• v.40 no.3
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.147-149
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• 2007

White-rot fungi which degrade lignin can also degrade diverse recalcitrant compounds such as polymeric dyes, explosives, pesticides, and endocrine disrupting chemicals. Lignin degrading enzymes are involved in the degradation reactions, and introduction of foreign genes into a white-rot fungus is required in order to increase the degrading capacity. Genetic transformation experiment has been carried out in Irpex lacteus and Phlebia tremellosa to an antibiotic resistance. The transformation yields were 50-70 transformants/${\mu}g$ DNA and 15-25 transformants/${\mu}g$ DNA in I. lacteus and P. tremellosa, respectively. The stable replication of the plasmid was confirmed by PCR using the plasmid-specific primers, and many mutants were generated during this integration in both fungi.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1932-1941
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• 2017

DFC-2, a methyl 5-[2-(dimethylamino)ethoxy]-7,12-dioxo-7,12-dihydrodinaphtho[1,2-b:2',3'-d]furan-6-carboxylate, is reported to have antitubercular effects against Mycobacterium tuberculosis. At concentrations ranging from 0.19 to $0.39{\mu}g/ml$, DFC-2 inhibited both drugusceptible and -resistant strains of M. tuberculosis. Microarray analyses were employed to gain insights into the molecular mechanisms of DFC-2's action in M. tuberculosis. The most affected functional gene category was "lipid biosynthesis," which is involved in mycolic acid synthesis. The decrease in transcription of genes related to mycolic acid synthesis was confirmed by RT-PCR. Furthermore, we found that DFC-2 triggered a reduction in mycolic acid levels, showing a similar pattern to that of mycolic acid synthesis inhibitor isoniazid. These results may explain how this compound kills mycobacteria efficiently by inhibiting mycolic acid synthesis.

• Nutrition Research and Practice
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• v.10 no.6
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• pp.623-628
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• 2016

BACKGROUND/OBJECTIVES: Obesity-induced steatohepatitis accompanied by activated hepatic macrophages/Kupffer cells facilitates the progression of hepatic fibrinogenesis and exacerbates metabolic derangements such as insulin resistance. Heme oxyganase-1 (HO-1) modulates tissue macrophage phenotypes and thus is implicated in protection against inflammatory diseases. Here, we show that the flavonoid quercetin reduces obesity-induced hepatic inflammation by inducing HO-1, which promotes hepatic macrophage polarization in favor of the M2 phenotype. MATERIALS/METHODS: Male C57BL/6 mice were fed a regular diet (RD), high-fat diet (HFD), or HFD supplemented with quercetin (HF+Que, 0.5g/kg diet) for nine weeks. Inflammatory cytokines and macrophage markers were measured by ELISA and RT-PCR, respectively. HO-1 protein was measured by Western blotting. RESULTS: Quercetin supplementation decreased levels of inflammatory cytokines ($TNF{\alpha}$, IL-6) and increased that of the anti-inflammatory cytokine (IL-10) in the livers of HFD-fed mice. This was accompanied by upregulation of M2 macrophage marker genes (Arg-1, Mrc1) and downregulation of M1 macrophage marker genes ($TNF{\alpha}$, NOS2). In co-cultures of lipid-laden hepatocytes and macrophages, treatment with quercetin induced HO-1 in the macrophages, markedly suppressed expression of M1 macrophage marker genes, and reduced release of MCP-1. Moreover, these effects of quercetin were blunted by an HO-1 inhibitor and deficiency of nuclear factor E2-related factor 2 (Nrf2) in macrophages. CONCLUSIONS: Quercetin reduces obesity-induced hepatic inflammation by promoting macrophage phenotype switching. The beneficial effect of quercetin is associated with Nrf2-mediated HO-1 induction. Quercetin may be a useful dietary factor for protecting against obesity-induced steatohepatitis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.3
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• pp.459-465
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• 2011

The nuclear factor of activated T cells (NFAT) protein induces transcriptions of cytokine genes including IL-2 for T-cell activation. Normally activation of NFAT is important to induce immune responses but excessive NFAT activation provokes immunopathological reactions such as autoimmunity, transplant rejection, and inflammation. Thus, for the treatment of autoimmune diseases drugs repressing the activation of NFAT have been searched. In this study, immnunosuppressive effects of Rosa chinensis Jacq. extracts identified as a potent NFAT inhibitor from a natural product library were examined. NFAT reporter assay, MTS assay, real time PCR, IL-2 ELISA, MLR, and FACS (Fluorescent Activated Cell Sorting) were used to measure inhibitory immunocyte activities of Rosa chinensis Jacq. The variety of natural products have been screened and some were found to show inhibitory activities against the NFAT transcription factor. Among them, extract of Rosa chinensis Jacq. showed an strong inhibitory effect on the activation of NFAT without affecting cell viability. Levels of IL-2 transcripts as well as IL-2 protein were decreased with treatment of Rosa chinensis Jacq. extract. In addition, immunosuppressive activity of Rosa chinensis Jacq. extract was exhibited in the mixed leukocytes reaction. The increasement of CD4+CD25+ (Treg) immunocyte was also detected in the analysis using FACS after applying Rosa chinensis Jacq. extract. Immunosuppressive effects of the Rosa chinensis Jacq. extracts were clearly demonstrated in the present study. In addition, Rosa chinensis Jacq. extract also positively affected regulatory T cell induction. Further investigations in particular on purification of single substance responsible for the immunosuppressive effects from the extract and analysis on possible actions of the extract in interfering cell signaling and cytokine production will be needed.

• Toxicological Research
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• v.33 no.3
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• pp.211-218
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• 2017

Cytochrome P450 1B1 (CYP1B1) acts as a hydroxylase for estrogen and activates potential carcinogens. Moreover, its expression in tumor tissues is much higher than that in normal tissues. Despite this association between CYP1B1 and cancer, the detailed molecular mechanism of CYP1B1 on cancer progression in HeLa cells remains unknown. Previous reports indicated that the mRNA expression level of Herc5, an E3 ligase for ISGylation, is promoted by CYP1B1 suppression using specific small interfering RNA, and that ISGylation may be involved in ubiquitination related to ${\beta}-catenin$ degradation. With this background, we investigated the relationships among CYP1B1, Herc5, and ${\beta}-catenin$. RT-PCR and western blot analyses showed that CYP1B1 overexpression induced and CYP1B1 inhibition reduced, respectively, the expression of $Wnt/{\beta}-catenin$ signaling target genes including ${\beta}-catenin$ and cyclin D1. Moreover, HeLa cells were treated with the CYP1B1 inducer $7,12-dimethylbenz[{\alpha}]anthracene$ (DMBA) or the CYP1B1 specific inhibitor, tetramethoxystilbene (TMS) and consequently DMBA increased and TMS decreased ${\beta}-catenin$ and cyclin D1 expression, respectively. To determine the correlation between CYP1B1 expression and ISGylation, the expression of ISG15, a ubiquitin-like protein, was detected following CYP1B1 regulation, which revealed that CYP1B1 may inhibit ISGylation through suppression of ISG15 expression. In addition, the mRNA and protein expression levels of Herc5 were strongly suppressed by CYP1B1. Finally, an immunoprecipitation assay revealed a direct physical interaction between Herc5 and ${\beta}-catenin$ in HeLa cells. In conclusion, these data suggest that CYP1B1 may activate $Wnt/{\beta}-catenin$ signaling through stabilization of ${\beta}-catenin$ protein from Herc5-mediated ISGylation for proteosomal degradation.

• Plant Biotechnology Reports
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• v.5 no.3
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• pp.217-224
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• 2011

Non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) is a popular vegetable in Asian countries. The diamondback moth (DBM), Plutella xylostella (L.), an insect with worldwide distribution, is a main pest of Brassicaceae crops and causes enormous crop losses. Transfer of the anti-insect gene into the plant genome by transgenic technology and subsequent breeding of insect-resistant varieties will be an effective approach to reducing the damage caused by this pest. We have produced transgenic non-heading Chinese cabbage plants expressing the potato proteinase inhibitor II gene (pinII) and tested the pest resistance of these transgenic plants. Non-heading Chinese cabbages grown for 45 days on which buds had formed were used as experimental materials for Agrobacterium-mediated vacuum infiltration transformation. Forty-one resistant plants were selected from 1166 g of seed harvested from the infiltrated plants based on the resistance of the young seedlings to the herbicide Basta. The transgenic traits were further confirmed by the Chlorophenol red test, PCR, and genomic Southern blotting. The results showed that the bar and pinII genes were co-integrated into the resistant plant genome. A bioassay of insect resistance in the second generation of individual lines of the transgenic plants showed that DBM larvae fed on transgenic leaves were severely stunted and had a higher mortality than those fed on the wild-type leaves.

• Molecules and Cells
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• v.38 no.5
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• pp.416-425
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• 2015

NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2- upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-$G_0/G_1$ peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

• Molecules and Cells
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• v.28 no.2
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• pp.125-130
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• 2009

Tacrolimus is a widely used T cell targeted immunosuppressive drug, known as a calcineurin inhibitor. However, the exact pharmacological effects of tacrolimus on $CD4^+$ T cells have yet to be elucidated. This study investigated the effects of tacrolimus on $CD4^+$ T cell subsets. Mouse or human $CD4^+$ T cells were cultured with immobilized anti-CD3/CD28 antibodies in the presence of tacrolimus. The cell division of $CD4^+$ T cells was analyzed using a flow cytometer according to the expression of Foxp3. The gene expression patterns of tacrolimus-exposed T cells were examined by quantitative PCR. In the case of conventional $CD4^+$ T cells (Tconv cells), tacrolimus inhibited T cell receptor stimulation-induced cell division. In contrast, the cell division of regulatory $CD4^+$ T cells (Treg cells) was even promoted in the presence of tacrolimus, especially in humans. Tacrolimus did not promote conversion of Tconv to Treg cells in mice. Furthermore, tacrolimus modified the expression levels of Foxp3-regulated T cell receptor signal related-genes, PTPN22 and Itk, in human Treg cells. Immunosuppressive effect of tacrolimus may be attributed to the relatively enhanced proliferation of Treg cells in association with altered gene expression levels of TCR signaling molecules.

• Molecules and Cells
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• v.39 no.12
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• pp.869-876
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• 2016

Cantharidin (CTD) is an active compound isolated from the traditional Chinese medicine blister beetle and displayed anticancer properties against various types of cancer cells. However, little is known about its effect on human chronic myeloid leukemia (CML) cells, including imatinib-resistant CML cells. The objective of this study was to investigate whether CTD could overcome imatinib resistance in imatinib-resistant CML cells and to explore the possible underlying mechanisms associated with the effect. Our results showed that CTD strongly inhibited the growth of both imatinib-sensitive and imatinib-resistant CML cells. CTD induced cell cycle arrest at mitotic phase and triggered DNA damage in CML cells. The ATM/ATR inhibitor CGK733 abrogated CTD-induced mitotic arrest but promoted the cytotoxic effects of CTD. In addition, we demonstrated that CTD downregulated the expression of the BCR-ABL protein and suppressed its downstream signal transduction. Real-time quantitative PCR revealed that CTD inhibited BCR-ABL at transcriptional level. Knockdown of BCR-ABL increased the cell-killing effects of CTD in K562 cells. These findings indicated that CTD overcomes imatinib resistance through depletion of BCR-ABL. Taken together, CTD is an important new candidate agent for CML therapy.