• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.532-541
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• 2010

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1925-1931
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• 2017

Korean red pine (Pinus densiflora) bark extract, PineXol (PX), was investigated for its potential antioxidant and anti-inflammation effects in vitro. It was hypothesized that PX treatment ($25-150{\mu}g/ml$) would reduce the lipid synthesis in HepG2 hepatocytes as well as lipid accumulation in 3T3-L1 adipocytes. Hepatocytes' intracellular triglycerides and cholesterol were decreased in the PX $150{\mu}g/ml$ treatment group compared with the control (p < 0.05). Consequently, de novo lipogenic proteins (acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, elongase of very long chain fatty acids 6, glycerol-3-phosphate acyltransferase 1, and sterol regulatory element-binding protein 1) were significantly decreased in hepatocytes by PX $150{\mu}g/ml$ treatment compared with the control (p < 0.05). In differentiated 3T3-L1 adipocytes, the lipid accumulation was significantly attenuated by all PX treatments (p < 0.01). Regulators of adipogenesis, including CCAAT-enhancer-binding proteins alpha, peroxisome proliferatoractivated receptor gamma, and perilipin, were decreased in PX $100{\mu}g/ml$ treatment compared with the control (p < 0.05). In conclusion, PX might have anti-obesity effects by blocking hepatic lipogenesis and by inhibiting adipogenesis in adipocytes.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.45-49
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• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.346-351
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• 1999

Tumor cells may alter the expression of proteins involved in antigen processing and presentation, allowing them to avoid recognition and elimination by cytotoxic T cells. In order to investigate whether the major histocompatibility complex (MHC) class I-mediated antigen processing machinery is preserved in human lung cancer cell lines, we examined the expression of multiple components of the MHC class I antigen processing pathway, including transporter associated with antigen processing (TAP), $\beta_2$-microglobulin, MHC class I molecules, and chaperones which have not been previously examined in this context. Row cytometry analysis showed that the cell surface expression of MHC class I molecules was downregulated in all of the cell lines. While some cell lines showed no detectable expression of MHC class I molecules, pulse-chase experiments showed that MHC class I molecules were synthesized in the other cell lines but not transported from the endoplasmic reticulum to the cell surface. Low or nondetectable levels of TAP1 and/or TAP2 expression were demonstrated by Western blot analysis in all of the cell lines, representing a variety of lung tissue types. In some cases, this was accompanied by loss of tapasin expression. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. This study provides further information for designing the potential therapeutic applications such as immunotherapy and gene therapy against cancers.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Molecules and Cells
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• v.39 no.2
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• pp.96-102
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• 2016

miR-101 is considered to play an important role in hepatocellular carcinoma (HCC), but the underlying molecular mechanism remains to be elucidated. Here, we aimed to confirm whether Girdin is a target gene of miR-101 and determine the tumor suppressor of miR-101 through Girdin pathway. In our previous studies, we firstly found Girdin protein was overexpressed in HCC tissues, and it closely correlated to tumor size, T stage, TNM stage and Edmondson-Steiner stage of HCC patients. After specific small interfering RNA of Girdin was transfected into HepG2 and Huh7.5.1 cells, the proliferation and invasion ability of tumor cells were significantly inhibited. In this study, we further explored the detailed molecular mechanism of Girdin in HCC. Interestingly, we found that miR-101 significantly low-expressed in HCC tissues compared with that in matched normal tissues while Girdin had a relative higher expression, and miR-101 was inversely correlated with Girdin expression. In addition, after miR-101 transfection, the proliferation, migration and invasion abilities of HepG2 cells were weakened. Furthermore, we confirmed that Girdin is a direct target gene of miR-101. Finally we confirmed Talen-mediated Girdin knockout markedly suppressed cell proliferation, migration and invasion in HCC while downregulation of miR-101 significantly restored the inhibitory effect. Our findings suggested that miR-101/Girdin axis could be a potential application of HCC treatment.

• Natural Product Sciences
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• v.17 no.3
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• pp.250-255
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• 2011

The immune system is finely balanced by the activities of pro-inflammatory and anti-inflammatory mediators or cytokines. Unregulated activities of these mediators can lead to the development of various inflammatory diseases. A variety of safe and effective anti-inflammatory agents are available with many more drugs under development. Of the natural compounds, the sesquiterpenes (nootkatone, ${\alpha}$-cyperone, valencene and ${\alpha}$-selinene) isolated from C. rotundus L. have received much attention because of their potential antiinflammatory effects. However, limited studies have been reported regarding the influence of sesquiterpene structure on anti-inflammatory activity. In the present study, the anti-inflammatory potential of four structurally divergent sesquiterpenes was evaluated in lipopolysaccaride (LPS)-stimulated RAW 264.7 cells, murine macrophages. Among the four sesquiterpenes, ${\alpha}$-cyperone and nootkatone, showed stronger anti-inflammatory and a potent NF-${\kappa}B$ inhibitory effect on LPS-stimulated RAW 264.7 cells. Molecular analysis revealed that various inflammatory enzymes (iNOS and COX-2) were reduced significantly and this correlated with downregulation of the NF-${\kappa}B$ signaling pathway. Additionally, electrophoretic mobility shift assays (EMSA) elucidated that nootkatone and ${\alpha}$-cyperone dramatically suppressed LPS-induced NF-${\kappa}B$-DNA binding activity using 32Plabeled NF-${\kappa}B$ probe. Hence, our data suggest that ${\alpha}$-cyperone and nootkatone are potential therapeutic agents for inflammatory diseases.

• Korean Journal of Pharmacognosy
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• v.39 no.2
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• pp.95-103
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• 2008

In the present study, we investigated the anti-inflammatory effects by kaempferol-3-O-${\beta}$-D-sophoroside (KS) isolated from Sophora japonica (Leguminosae) on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin ($PGE_2$) production by RAW 264.7 cell line compared with kaempferol. KS significantly inhibited the LPS-induced NO and $PGE_2$ production. Consistent with these observations, KS reduced the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner. In addition, the release and the mRNA expression levels of tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and interleukin-6 (IL-6) were also reduced by KS. Moreover, KS attenuated the LPS-induced activation of nuclear factor-kappa B ($NF{-\kappa}B$), a transcription factor necessary for pro-inflammatory mediators, iNOS, COX-2, $TNF-{\alpha}$ and IL-6 expression. These results suggest that the down regulation of iNOS, COX-2, $TNF-{\alpha}$, and IL-6 expression by KS are achieved by the downregulation of $NF{-\kappa}B$ activity, and that is also responsible for its anti-inflammatory effects.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.1
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• pp.155-161
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• 2008

Carbon tetrachloride $(CCl_4)-induced$ liver injury depends on a toxic agent that has to be metabolized by the liver NAPDH-cytochrome P450 enzyme system to a highly reactive intermediate. Although several isoforms of cytochrome P450 may metabolize $CC1_4$, attention has been focused largely on the cytochrome P450 2E1 (CYP2E1), which is ethanol-inducible. Alternations in the activity of CYP2E1 affect the susceptibility to hepatic injury from $CC1_4$. In this study, the liver protective effect of the hot water extracts of Scutellaria radix (SR) was investigated. The SR exhibited a hepatoprotective activity against $CCl_4-induced$ liver damage in Chang liver cells. The expression of CYP2E1, measured by RT-PCR and Western blot analysis, was significantly decreased by SR treatment in Chang cells. Based on these findings, it is suggested that hepatoprotective effect of SR possibly related to downregulation of CYP2E1 expression.