• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.280-288
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• 2024

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.3
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• pp.209-217
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• 2007

Purpose: Multidrug resistance (MDR) of the cancer cells related to mdr1 gene expression can be effectively treated by selective short hairpin RNA for mdr1 gene (shMDR). Sodium/iodide symporter (NIS) gene is well known to have both reporter and therapeutic gene characteristics. We have co-transfected both shMDR and NIS gene into colon cancer cells (HCT15 cell) expressing MDR and Tc-99m sestamibi and I-125 uptake were measured. In addition, cytotoxic effects of doxorubicin and I-131 therapy were also assessed after transfection. Material and Methods: At first, shMDR was transfected with liposome reagent into human embryonic kidney cells (HEK293) and HCT cells. shMDR transfection was confirmed by RT-PCR and western blot analysis. Adenovirus expressing NIS (Ad-NIS) gene and shMDR (Ad-shMDR) were co-transfected with Ad-NIS into HCT15 cells. Forty-eight hours after infection, inhibition of P-gycoprotein (Pgp) function by shMDR was analyzed by a change of Tc-99m sestamibi uptake and doxorubicin cytotoxicity, and functional activity of induced NIS gene expression was assessed with I-125 uptake assay. Results: In HEK293 cells transfected with shMDR, mdr1 mRNA and Pgp protein expressions were down regulated. HCT15 cells infected with 20 MOI of Ad-NIS was higher NIS protein expression than control cells. After transfection of 300 MOI of Ad-shMDR either with or without 10 MOI of Ad-NIS, uptake of Tc-99m sestamibi increased up to 1.5-fold than control cells. HCT15 cells infected with 10 MOI of Ad-NIS showed approximately 25-fold higher I-125 uptake than control cells. Cotransfection of Ad-shMDR and Ad-NIS resulted in enhanced cytotoxic by doxorubicin in HCT15 cells. I-131 treatment on HCT15 cells infected with 20 MOI of Ad-NIS revealed increased cytotoxic effect. Conclusion: Suppression of mdr1 gene expression, retention of Tc-99m sestamibi, enhanced doxorubicin cytotoxicity and increases in I-125 uptake were achieved in MDR expressing cancer cell by co-transfection of shMDR and NIS gene. Dual therapy with doxorubicin and radioiodine after cotransfection shMDR and NIS gene can be used to overcome MDR.

• Journal of Life Science
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• v.26 no.8
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• pp.875-880
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• 2016

The plasma membrane plays a crucial role in relaying signals from the outside environment to the inside of the cells. In eukaryotic cells, the inner leaflets of the plasma membrane are composed mostly of phospholipids, including phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositides (PIs). In this study, we tried to analyze the morphological changes induced by EGFP-fused membrane binding proteins, which are targeted to the plasma membrane via specific phospholipids binding. As a result, we found that overexpression of EGFP-P4M-SidM, a specific PI4P binding protein, or EGFP alone, did not induce any morphological changes. On the other hand, overexpression of EGFP-PLCδ1(PH), which is a specific PI(4,5)P₂ binding protein, EGFP-AKT1(PH) which binds to PI(3,4,5)P₃, or EGFP-OSH2(PH)×2 which binds to PI4P and PI(4,5)P₂, could induce the filopodia and lamilapodia formation as well as cell shrinkage. Overexpression of Lact-C2-EGFP which is a specific PS-binding probe, EGFP fused Aplysia phosphodiesterase 4 (ApPDE4) long-form (L(N20)-EGFP) which is localized to the plasma membrane via hydrophobic interaction, or EGFP fused Aplysia PDE4 short-form (S(N-UCR1-2)-EGFP) which is localized to the plasma membrane via electrostatic interaction, could induce cell shrinkage, but not filopodia or lamilapodia formation. Taken together, our data support that the different phospholipid bindings in the plasma membrane could induce different characteristic morphological changes. Thus, we can analyze, characterize, and classify the cellular morphological changes induced by the various phospholipid binding proteins.

• Korean Journal of Agricultural Science
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• v.47 no.1
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• pp.83-93
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• 2020

Lactoferrin (LF) is an iron-binding glycoprotein that is present in colostrum, milk, and other body secretions. The objective of this study was to investigate the effects of lactoferrin hydrolysates (LHs) on the production of immunomodulatory factors, including inflammatory related cytokines. The nuclear factor (NF)-κB reporter assay using human embryonic kidney 293 cells (HEK-293) revealed that NF-κB activity was significantly decreased by 1, 50, and 100 ㎍/mL of LH and the fractions above and below the 10 kDa LH. The mRNA expression of interferon (IFN)-γ in rat basophilic leukemia mast cells (RBL-2H3) treated with the fraction above the 10 kDa LH decreased in a dose-dependent manner, but the cells treated with LH and the fraction below the 10 kDa LH showed an increased expression of IFN-γ in a dose-dependent manner. The level of cyclooxygenase (COX)-2 expression decreased dose-dependently in RBL-2H3 cells treated with LH and the fraction above the 10 kDa LH, but the cells treated with the fraction below the 10 kDa LH showed an increased COX-2 expression in a dose-dependent manner. The mRNA expression of interleukin (IL)-4) was dose-dependently decreased by the fraction below the 10 kDa LH in human mast cells (HMC-1). The mRNA expressions of tumor necrosis factor (TNF)-α and IL-6 were significantly dose-dependently decreased by the fractions above and below the 10 kDa LH, but was dose-dependently increased by LH. The production of IL-4 was a little increased by the fraction above the 10 kDa LH compared to the positive control, but was decreased with LH and the fraction below the 10 kDa LH in HMC-1 cells. It was concluded that LF hydrolysates had an immunomodulating effect on anti-, pro-inflammatory and anti-allergic reactions.

• BMB Reports
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• v.36 no.3
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• pp.275-281
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• 2003

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.47-47
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• 2003

The presenilin 1 (PS1) or PS2 is an essential component of the ${\gamma}$-secretase complex, which mediates the intramembrane proteolysis of selected type-I membrane, including the ${\beta}$-amyloid precursor protein (APP) to yield A${\beta}$. Familial Alzheimer's disease (FAD)-associated mutations in presenilins give rise to an increased production of a highly amyloidogenic A${\beta}$42. In addition to their well-documented proteolytic function, the presenilins play a role in calcium signaling. We have previously reported that presenilin FAD mutations cause highly consistent alterations in intracellular calcium signaling pathways, which include deficits in capacitative calcium entry (CCE), the refilling mechanism for depleted internal calcium stores. However, molecular basis for the presenilin-mediated modulation of CCE remains to be elucidated. In the present study, whole-cell patch clamp method was used to identify a specific calcium-permeable ion channel current(s) that is responsible for the CCE deficits associated with FAD-linked PS1 mutants. Unexpectedly, both voltage-activated and conventional store depletion-activated calcium currents I(CRAC), were absent in HEK293 cells, which were stably transfected either with wild-type or FAD mutant (L286V, M146L, and delta E9) forms of PS1. Recently, magnesium-nucleotide-regulated metal cation current, or I(MagNum), has been described and appears to share many common properties with I(CRAC) including calcium permeability and inhibitor sensitivity (e.g. 2-APB). We have detected I(MagNum) in all 293 cells tested. Interestingly, FAD mutant 293 cells developed only about half of currents compared to PS1 wild type cells.

• BMB Reports
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• v.49 no.6
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• pp.319-324
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• 2016

RNA polymerase II C-terminal domain phosphatases are newly emerging family of phosphatases that contain FCPH domain with Mg⁺²-binding DXDX(T/V) signature motif. Its subfamily includes small CTD phosphatases (SCPs). Recently, we identified several interacting partners of human SCP1 with appearance of dephosphorylation and O-GlcNAcylation. In this study, using an established cell line with inducible CTDSPL2 protein (a member of the new phosphatase family), proteomic screening was conducted to identify binding partners of CTDSPL2 in nuclear extract through immunoprecipitation of CTDSPL2 with its associated. This approach led to the identification of several interacting partners of CTDSPL2. This will provide a better understanding on CTDSPL2.

• Biomedical Science Letters
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• v.17 no.3
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• pp.185-190
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• 2011

Adenovirus type 5 (Ad5) vectors have been used for gene transfer to a wide variety of cell types in vivo and in vitro. The advantages of adenovirus vectors include the high titer of virus readily obtained in large scale preparations, their ability to transduce dividing and non dividing cells, and the high level of transgene expression. Since adenovirus vectors do not integrate in host cell DNA, there is a lack of insertional mutagenesis. However, many human tumor cells lack expression of the adenovirus 5 receptors and contain areas of hypoxia. In order to identify the pattern of replication and gene expression of oncolytic adenovirus in hypoxic condition, multiple different fiber modified Ads (Ad5F/S11, Ad5F/S35, Ad5F/K7, Ad5F/K21, and Ad5F/RGD) was compared. The replication of all fiber modified adenovirus was inhibited in hypoxic condition in HEK 293 cells, but gene expression has variety on different tumor cell lines and the level of coxackievirus and adenovirus receptor (CAR) expression. These data suggest that CAR expression pattern and hypoxic condition of tumor are considered for optimal oncolytic adenovirus application.

• Journal of the Korean Applied Science and Technology
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• v.37 no.4
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• pp.674-681
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• 2020

The root of Codonopsis lanceolata has been used in traditional medicine. This study was conducted to confirm the comparative effect of ethanol solvent extraction (CLE) and supercritical carbon dioxide extraction (CLS) of C. lanceolata roots. CLS had higher antioxidant than CLE. For supercritical co-solvent modified carbon dioxide extraction (CLS), it were extracted at 250 bar 50℃ 150 min at a flow rate of ethyl alcohol 3 mL/min for 90min. In addition, CLS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 ㎍/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells increased approximately by 3-folds compared to that of the untreated control group. Also, the treatment by CLS (50 ㎍/mL) showed a significant increase of involucrin expression. These results indicate that supercritical carbon dioxide extract of C. lanceolatamay serve as a cosmeceutical agent for improving skin barrier function and the treatment of obesity.

• Journal of the Korean Applied Science and Technology
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• v.36 no.4
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• pp.1136-1142
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• 2019

The root of Paeonia lactiflora has been used in Chinese medicine. We conducted to check the comparative qualities of ethanol solvent extraction (PLE) and supercritical carbon dioxide extraction (PLS) of P. lactiflora root. PLE had higher antioxidant and polyphenol contents than PLS. But, PLS were significantly increased peroxisome proliferator-activated receptor (PPAR)-α. In addition, PLS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 ㎍/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells increased approximately by 3-folds compared to that of the untreated control group. These results indicate that P. lactiflora supercritical carbon dioxide extract may serve as a cosmeceutical for improving skin barrier function and the treatment of obesity.