• YAKHAK HOEJI
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• v.49 no.3
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Natural Product Sciences
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• v.13 no.2
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• pp.123-127
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• 2007

We investigated effect of small black soybean fraction (SBSF) T cell-mediated responses for tumor surveillance and proliferation in leukemia cells in vitro. Each SBSF butanol fraction (SBSFBu) and SBSF chloroform fraction (SBSFCh) was administered p.o. once a day far 21 days in BALB/c mice and then levels of serum cytokines and subpopulation of lymphocytes were measured. Moreover, SBSF fraction was treated into the cultured various cell lines for proliferation in leukemia cell lines, NO production by RAW264.7 cells, and expression of p53 gene in U937 leukemia cells. These results showed that SBSFBu increased levels of serum IL-4but not IL-2 and IFN-${\gamma}$, and increased expression of CD4$^+$ T cells and CD8$^+$ T cells in splenocytes in vivo, while SBSFCh increased levels of serum IL-2 and IFN-${\gamma}$ but decreased IL-4, and increased CD8$^+$ T cells but not CD4$^+$ T cells. Moreover, both of SBSFBu and SBSFCh inhibited proliferation of HL60, U937, and L1210 leukemia cell lines in a dose-dependent manner, up-regulated NO production by RAW264.7 cells in a dose-dependent manner, and enhanced expression of p53 gene in U937 leukemia cells. Our findings indicate that SBSFBu and SBSFCh may enhance T cell-dependent immune responses, and that both of SBSFBu and SBSFCh may inhibit proliferation of leukemia cells by up-regulation of NO production and expression of p53 gene.

• Animal Bioscience
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• v.36 no.2
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• pp.295-306
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• 2023

Objective: Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Methods: Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. Results: The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. Conclusion: p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.

• Journal of the Korean Society of Food Science and Nutrition
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• v.38 no.12
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• pp.1685-1690
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• 2009

In this study, we investigated the effect of C. zawadskii extract on nitric oxide (NO) production, prostaglandin E2 (PGE2) production, protein and mRNA expression of inducible nitric oxide synthase (iNOS) in LPS-induced RAW 264.7 macrophage cells. C. zawadskii extract (5~50 μg/mL) significantly inhibited LPS-induced NO production in a concentration-dependent manner ranging from 23.3% to 100%. Consistent with the inhibitory effect on NO production, C. zawadskii extract inhibited the protein expression and mRNA expression of iNOS. Although flower extracts of C. zawadskii was not effective on the expression of PGE2 and COX-2, flower extracts of C. zawadskii, however, showed a strong anti-inflammatory activity through inhibition of NO production and iNOS expression. The present results suggest that C. zawadskii extract has an inhibitory effect on NO production, and thus can be used as an anti-inflammatory agent.

• Journal of Nutrition and Health
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• v.54 no.5
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• pp.435-447
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• 2021

Purpose: Body adiposity is negatively correlated with hepatic iron status, and Korean pine nut oil (PNO) has been reported to reduce adiposity. Therefore, we aimed to study the effects of PNO on adiposity, hepatic mineral status, and the expression of genes and proteins involved in iron absorption. Methods: Five-week-old male C57BL/6 mice were fed a control diet containing 10% kcal from PNO (PC) or soybean oil (SBO; SC), or a high-fat diet (HFD) containing 35% kcal from lard and 10% kcal from PNO (PHFD) or SBO (SHFD). Hepatic iron, copper, and zinc content; and expression of genes and proteins related to iron absorption were measured. Results: HFD-fed mice had a higher white fat mass (2-fold; p < 0.001), lower hepatic iron content (25% lower; p < 0.001), and lower hepatic Hamp (p = 0.028) and duodenal Dcytb mRNA levels (p = 0.037) compared to the control diet-fed mice. Hepatic iron status was negatively correlated with body weight (r = -0.607, p < 0.001) and white fat mass (r = -0.745, p < 0.001). Although the PHFD group gained less body weight (18% less; p < 0.05) and white fat mass (18% less; p < 0.05) than the SHFD group, the hepatic iron status impaired by the HFD feeding did not improve. The expression of hepatic and duodenal ferroportin protein was not affected by the fat amount or the oil type. PNO-fed mice had significantly lower Slc11a2 (p = 0.022) and Slc40a1 expression (p = 0.027) compared to SBO-fed mice. However, the PC group had a higher Heph expression than the SC group (p < 0.05). The hepatic copper and zinc content did not differ between the four diet groups, but hepatic copper content adjusted by body weight was significantly lower in the HFD-fed mice compared to the control diet-fed mice. Conclusion: HFD-induced obesity decreased hepatic iron storage by affecting the regulation of genes related to iron absorption; however, the 18% less white fat mass in the PHFD group was not enough to improve the iron status compared to the SHFD group. The hepatic copper and zinc status was not altered by the fat amount or the oil type.

• Food Science and Biotechnology
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• v.17 no.1
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• pp.106-113
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• 2008

The antioxidant and anti-inflammatory protective effects of $\beta$-glucan from barley on RAW 264.7 murine macrophage cells induced by lipopolysaccharide (LPS) were examined. The RAW 264.7 murine macrophages were preincubated with various concentrations ($0-200\;{\mu}g/mL$) of $\beta$-glucan and stimulated with LPS to induce oxidative stress and inflammation. The $\beta$-glucan treatments were found to reduce thiobarbituric acid-reactive substance (TBARS) accumulation, and enhance glutathione levels and the activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase, glutathione reductase, and glutathione peroxidase (GSH-px) in the LPS-stimulated macrophages as compared to the LPS-only treated cells. Nitric oxide (NO) production was significantly suppressed in a dose-dependent manner (p<0.05) with an $IC_{50}$ of $104\;{\mu}g/mL$. Further treatment with $\beta$-glucan at $200\;{\mu}g/mL$ suppressed NO production to 2% of the LPS-control, and suppressed the levels of inducible nitric oxide synthase (iNOS) protein and mRNA in a dose-dependent manner. The specific DNA binding activity of nuclear factor ${\kappa}B\;(NF{\kappa}B)$ was significantly suppressed by $\beta$-glucan treatment with an $IC_{50}$ of $220\;{\mu}g/mL$ in a dose-dependent manner. Finally, barley $\beta$-glucan ameliorates NO production and iNOS expression through the down-regulation of $NF{\kappa}B$ activity, which may be mediated by attenuated oxidative stress in RAW 264.7 macrophages.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.106-115
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• 2010

Phosphorylated dextran (P-Dex) is an acidic polysaccharide that functions as an immune adjuvant. P-Dex is known to regulate immune response by maintaining a balance between Th1 and Th2 cells in vitro, and thus may also be important in the control of allergic reactions. In the current study, we report the optimum conditions required for the efficient phosphorylation of dextran without toxicity. We found that when dextran was heated at 160${^{\circ}C}$ for 24 h in phosphate buffer (pH 5.0), the resulting P-Dex demonstrated the highest phosphorus content (6.8%). We also report that P-Dex enhances mitogenic activity in mouse splenocytes and induces expression of CD69 and CD86 on the surface of B cells and dendritic cells (DC) in vitro. Oral administration of P-Dex to ovalubmin (OVA)-immunized mice was found to reduce antigen-induced cell proliferation and suppress the expression of CD86 on Th2-inducing DC via exogenous OVA stimulation. P-Dex was also found to increase IL-10 expression in the splenocytes of treated mice. These findings suggest that oral administration of P-Dex increases immunological tolerance and improves the specificity of immunological response to specific antigens.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.975-979
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• 2006

The extracts of Oenanthe javanica were evaluated for their effects on the expression of cyclooxygenase-2 (COX-2), which is mediated by the translocation of the p65 subunit into the nucleus. Fractions of ethyl acetate and chloroform from 80% ethanol extracts of O. javanica exhibited inhibitory effects on the secretion of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) from lipopolysaccharide (LPS)-stimulated peritoneal macrophages; however, the aqueous- and hexane-fractions showed no significant effect. The ethyl acetate- and chloroform-fractions also reduced the COX-2 enzyme levels after 24-hr treatment. RT-PCR showed that the mRNA levels of COX-2 decreased following treatment with these fractions, suggesting that COX-2 expression is transcriptionally regulated by these extracts. We examined the effects of the chloroform- and ethyl acetate-fractions on the cytosolic activation of nuclear factor-${\kappa}B$ ($NF-{\kappa}B$, p65 subunit) and on the degradation of inhibitor-${\kappa}B{\alpha}$ ($I-{\kappa}B{\alpha}$) in order to determine the mechanism of COX-2 regulation. The LPS-stimulated activation of the p65 subunit was significantly blocked upon the addition of $50\;{\mu}g/mL$ of these fractions, and the cytosolic $I-{\kappa}B{\alpha}$ degradation process was simultaneously inhibited. These findings suggest that the inhibition of COX-2 expression by the ethyl acetate-and chloroform-fractions may result from the inhibition of p65 translocation by blocking the degradation of $I-{\kappa}B{\alpha}$; this may be the mechanistic basis for the anti-inflammatory effects of O. javanica.

• Molecular & Cellular Toxicology
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• v.3 no.3
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• pp.165-171
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• 2007

Mitomycin C (MMC), an antitumor antibiotic isolated from Streptomyces caespitosus, is used in chemotherapy of gastric, bladder and colorectal cancer. MMC is activated in vivo to alkylate and crosslink DNA, via G-G interstrand bonds, thereby inhibiting DNA synthesis and transcription. This study investigates gene expression changes in response to MMC treatment in order to elucidate the mechanisms of MMC-induced toxicity. MMC was admistered with single dose (0.32 and 1.6 ${\mu}M$) to TK6 cells. Applied Biosystem's DNA chips were used for identifying the gene expression profile by MMC-induced toxicity. We identified up- or down-regulated 90 genes including cyclin M2, cyclin-dependent kinase inhibitor 1A (p21, cip1), programmed cell death 1, tumor necrosis factor (ligand) superfamily, member 9, et al. The regulated genes by MMC associated with the biological pathways apoptosis signaling pathway. Further characterization of these candidate markers related to the toxicity will be useful to understand the detailed mechanism of action of MMC.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.107-116
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• 2019

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin's but not resveratrol's anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.