• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Journal of Life Science
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• v.30 no.12
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• pp.1092-1100
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• 2020

The six-transmembrane epithelial antigen of prostate 4 (Steap4) is a metalloreductase that plays a role in intracellular iron and cupper homeostasis, inflammatory response, and glucose and lipid metabolism. Previously, Steap4 has been reported to stimulate adipocyte differentiation; however, the underlying mechanisms of this action remain unexplored. In the present study, we investigated the molecular mechanisms involved in Steap4-induced adipocyte differentiation using 3T3-L1 cells, immortalized brown adipocyte (iBA) cells, and mouse embryonic fibroblast C3H10T1/2 cells. The knockdown of Steap4 using adenovirus-containing shRNA attenuated mitotic clonal expansion (MCE), as evidenced by the impaired proliferation of 3T3-L1 cells, iBA cells, and C3H10T1/2 cells within 48 hr after adding the differentiation medium. Steap4 knockdown downregulated G1/S phase transition-related cell cycle regulators (including cyclin A and cyclin D) and upregulated cell cycle inhibitors (including p21 and p27). Furthermore, Steap4 knockdown inhibited the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and Akt. Moreover, Steap4 knockdown repressed the expression of early adipogenic activators, such as CCAAT-enhancer-binding protein β (C/EBPβ) and Kruppel-like factor family factor 4 (KLF4). On the other hand, Steap4 knockdown stimulated the expression of adipogenic inhibitors, including KLF2, KLF3, and GATA2. The overexpression of Steap4 using an adenovirus removed the repressive histone marks H3K9me2 and H3K9me3 on the promoter of C/EBPβ. These results indicate that Stepa4 stimulates adipocyte differentiation through the induction of MCE and the modulation of early adipogenic transcription factors, including C/EBPβ, during the early phase of adipocyte differentiation.

• Tuberculosis and Respiratory Diseases
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• v.44 no.1
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• pp.162-174
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• 1997

Background : Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hyporthesized that treatment of tumor cells with retinoic acid could augment the bystander effect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. Methods : We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gene to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of tumor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. Results : Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin 43 and was asspciated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no delectable connexin 43. Conclusion : These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.

• Tuberculosis and Respiratory Diseases
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• v.63 no.3
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• pp.235-241
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• 2007

Background: Airway mucus hypersecretion plays an important role in the pathogenesis of asthma, and is associated with the induction of MUC5AC expression in airway secretion. The MUC5AC gene is highly polymorphic; however, there are few studies about the association between the polymorphisms of the MUC5AC gene and asthma susceptibility or asthma phenotypes. We have investigated the association of MUC5AC promoter polymorphisms with the risk of asthma and asthma phenotypes. Methods: We determined the genotypes of the MUC5AC promoter (-1274G>A) in 78 asthma patients and in 78 age, sex-matched control individuals in the Korean population. Genomic DNAs from blood were analyzed by PCR and RFLP (restriction fragment length polymorphism) determination. We examined $FEV_1$, total eosinophil count, serum IgE level, $PC_{20}$ and the presence of atopy (by a skin test) in asthma patients. Results: The mean age of the patients was $47.7{\pm}16.1$ years and 38.5% were men, and the mean $FEV_1$ was $84.4{\pm}22.3%$ of predicted in the asthma patients. The -1274G>A polymorphism of the MUC5AC promoter in asthma patients was not significantly different as compared with normal individuals (GG 57.7%, AG 34.6% and AA 7.7% in asthma patients vs. GG 56.4%, AG 38.5% and AA 5.1% in control subject, p = 0.752, Cod). Several clinical parameters in asthma patients such as $FEV_1$, total eosinophil count, serum IgE level, $PC_{20}$ and the presence of atopy, were not associated with the -1274G>A polymorphism of the MUC5AC promoter. Conclusion: The -1274G>A single nucleotide polymorphism (SNP) frequency of the MUC5AC promoter was not associated with asthma in a Korean population.

• Radiation Oncology Journal
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• v.21 no.3
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• pp.216-221
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• 2003

Purpose: To investigate the modulation of radiosensitivity by celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on cancer cells over- and under-expressing COX-2. Materials and Methods: A clonogenic radiation survival analysis was performed on A549 human lung and MCF-7 human breast cancer cell lines incubated in both 1 and $10\%$ fetal bovine serum (FBS) containing media. The apoptosis in both cell lines was measured after treatment with radiation and/or celecoxib. Results: Celecoxib enhanced the radiation sensitivity of the A549 cells in the medium containing the $10\%$ FBS, with radiation enhancement ratios of 1.58 and 1.81 respectively, at surviving fractions of 0.1, with $30\muM\;and\;50\muM$ celecoxib. This enhanced radiosensitivity disappeared in the medium containing the $1\%$ FBS. Celecoxib did not change the radiation sensitivity of the MCF-7 cells in either media. The induction of apoptosis by celecoxib and radiation was not synergistic in either cell line. Conclwsion: Celecoxib, a selective COX-2 inhibitor, preferentially enhanced the effect of radiation on COX-2 over-expressing cancer cells compared to the cells with a low expression, and this effect disappeared on incubation of the cells during drug treatment in the medium with suboptimal serum concentration. Apoptosis did not appear to be the underlying mechanism of this radiation enhancement effect due to celecoxib on the A549 cells. These findings suggest radiosensitization by a selective COX-2 inhibitor is COX-2 dependent.

• Journal of Life Science
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• v.18 no.8
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• pp.1023-1030
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• 2008

NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.6
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• pp.813-819
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• 2010

The intracellular lipid droplets were stained with Oil Red O dye and quantified. Compared to the control, lipid accumulation was significantly decreased by 19.4% with the treatment of LCM at the concentration of $1000\;{\mu}g$/mL. Intracellular triglyceride (TG) level was also reduced by 21% at the concentration of $1000\;{\mu}g$/mL. To determine the mechanism for the reduction in TG content, levels of glucose uptake and glycerol release were measured. Incubation of the 3T3-L1 adipocytes with LCM did not affect the cellular uptake of glucose. However, the level of free glycerol released into the cultured medium drastically increased by 24.3% with the treatment of LCM. In subsequent measurements using quantitative real-time PCR, mRNA levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) except lipoprotein lipase (LPL) were significantly elevated at higher concentration. These results suggest that LCM partially stimulates the lipolysis through the induction of HSL and/or ATGL gene expression, resulting in the reduced lipid accumulation and increased glycerol release.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.11
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• pp.1688-1694
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• 2014

Jeju Hallabong Tangor (Citrus kiyomi${\times}$ponkan) is a Citrus species with a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated the anti-obesity effects of Hallabong Tangor peel extracts before (HLB) and after (HLB-C) bioconversion with cytolase based on modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 adipocytes. Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and increased flavanone aglycone forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with 0.5 mg/mL of Sinetrol (a positive control), HLB or HLB-C. Adipocyte differentiation was inhibited in both citrus groups, but not in control and Sinetriol groups. HLB and HLB-C tended to reduce insulin-induced mRNA levels of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$) and sterol regulatory element-binding protein 1c (SREBP1c). Compared to the control and Sinetrol groups, HLB and HLB-C markedly suppressed insulin-induced protein expression of $C/EBP{\alpha}$ and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$). The HLB and Sinetrol groups, but not HLB-C group, significantly increased adipolytic activity with higher release of free glycerol compared to the control group in differentiated 3T3-L1 adipocytes. These results suggest that bio-conversion of Hallabong Tangor peel extracts with cytolase increases aglycone flavonoids. Irrespective of bioconversion, both Hallabong Tangor peel extracts exert anti-obesity effects that may contribute to prevention of obesity through inhibition of adipocyte differentiation or induction of adipolytic activity.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1191-1196
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• 2007

Eugenol (4-allyl-2-methoxyphenol) is a main component of essential oils obtained from various spices. Recent reports have shown that eugenol induces growth inhibition and apoptosis of malignant tumor cells. In this study, the stimulatory effect of eugenol on cell differentiation was investigated in HL-60 promyelocytic leukemia cells. When HL-60 cells were treated in combination with 150 ${\mu}M$ of eugenol and 3 nM of $1{\alpha},25-dihydroxyvitamin$ $D_{3}$, cell growth was slower than that of cells treated with eugenol or $1{\alpha},25-dihydroxyvitamin$ $D_{3}$ alone. Eugenol enhanced low dose of $1{\alpha,25-dihydroxyvitamin }$ $D_{3}-induced$ a $G_{0}/G_{1}$ phase arrest in cell cycle. Consistent with this, combined treatment of eugenol and $1{\alpha},25-dihydroxyvitamin$ $D_{3}$ cooperatively increased p27 level and decreased cyclin A, cdk 2 and cdk 4 levels, which are cell cycle regulators related to $G_{0}/G_{1}$ arrest. According to flow cytometric analysis, the expression of CD14 (monocytic differentiation marker) was more increased in the cells co-treated with eugenol and $1{\alpha},25-dihydroxyvitamin$ $D_{3}$. These results indicate that eugenol potentiates cell differentiation mediated by $1{\alpha},25-dihydroxyvitamin$ $D_{3}$ of suboptimal concentration. The differentiation-inducing property of eugenol maybe contributes to chemopreventive activity of cancer.

• Development and Reproduction
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• v.12 no.1
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• pp.31-39
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• 2008

Neural tissue has limited intrinsic capacity of repair after injury, and the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesechymal-like stem cells from human adipose tissues (AT-MSCs), and studied on transdifferentiation-promoting conditions in neural cells. Dopaminergic and cholinergic neuron induction of AT-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulphoxide (DMSO) and butylated hydroxyanisole(BHA) in N2 Medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. AT-MSCs treated with bFGF, SHH and FGF8 were differentiatied into dopaminergic neurons that were immunopositive for TH antibody. Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor (bFGF), retinoic acid (RA) and sonic hedgehog (Shh). AT-MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including neuro D1, $\beta$-tubulin III, GFAP and nestinwas markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after preinduction medium culture, we confirmed the differentiation of dopaminergic and cholinergic neurons by TH/$\beta$-tubulin III or ChAT/ $\beta$-tubulin III positive cells. Conclusively, AT-MSCs can be differentiated into dopaminergic and cholinergic neuronsand these findings suggest that AT-MSCs are alternative cell source of treatment for neurodegenerative diseases.