• Journal of Interdisciplinary Genomics
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• v.5 no.2
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• pp.35-41
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• 2023

Background: Ca²⁺ signaling plays a vital role in neuronal signaling and altered Ca²⁺ homeostasis in Parkinson's disease (PD). Overexpression of αSYN significantly promote the Ca²⁺-Calmodulin (CaM) activity and subsequent nuclear translocation of nuclear factor of activated T cells (NFAT) transcription factor in dopaminergic neurons of midbrain. However, the exact role of Ca²⁺-CaM and NFAT in PD pathology is yet to be elucidated. Methods: We designed the CaM-NFAT-oligodeoxynucleotide (ODN), a synthetic short DNA containing complementary sequence for NFAT transcription factor and CaM mRNA. Then, the effect of CaM-NFAT-ODN on 1-methyl-4-phenylpyridinium (MPP⁺)-mediated neurotoxicity was investigated in mimic PD model in vitro. Results: First, the expression of αSYN and CaM was strongly increased in substantia nigra (SN) of PD and the expression of tyrosine hydroxylase (TH) was strongly increased in control SN. Additionally, the expression of apoptosis marker proteins was strongly increased in SN of PD. Transfection of CaM-NFAT-ODN repressed CaM and pNFAT, the target genes of this ODN in rat embryo primary mesencephalic neurons. It also reduced ERK phosphorylation, a downstream target of these genes. These results demonstrated that CaM-NFAT-ODN operated successfully in rat embryo primary mesencephalic neurons. Transfection of CaM-NFAT-ODN repressed TH reduction, αSYN accumulation, and apoptosis by MPP⁺-induced neurotoxicity response through Ca²⁺ signaling and mitogen-activated protein kinases (MAPK) signaling. Conclusion: Synthetic CaM-NFAT-ODN has substantial therapeutic feasibility for the treatment of neurodegenerative diseases.

• The Korea Journal of Herbology
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• v.27 no.2
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• pp.53-59
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• 2012

Objectives : This study was undertaken to investigate the effects of the GGEx18 ethyl acetate fraction (EF) on lipid accumulation and gene expression of fatty acid-oxidizing enzymes using 3T3-L1 adipocytes, C2C12 skeletal muscle cells, and NMu2Li liver cells. Methods : PPAR${\alpha}$, AMPK and UCPs transactivation was examined in NMu2Li hepatocytes, C2C12 myocytes, and 3T3-L1 preadipocytes using transient transfection assays. Results : 1. Compared with control, EF significantly increased the mRNA expression of VLCAD in 3T3-L1 adipocytes. 2. Compared with control, EF (0.1 ${\mu}g/ml$) significantly inhibited lipid accumulation in 3T3-L1 adipocytes. 3. EF significantly increased the mRNA expression of AMPK${\alpha}$1, AMPK${\alpha}$2 and PPAR${\alpha}$ in C2C12 skeletal muscle cells compared with control. 4. EF significantly increased the mRNA expression of genes involved in fatty acid ${\beta}$-oxidation, such as thiolase, MCAD, and CPT-1 in C2C12 skeletal muscle cells compared with control. 5. EF significantly increased the mRNA expression of UCP2 involved in energy expenditure in C2C12 skeletal muscle cells compared with control. 6. Compared with control, EF (10 ${\mu}g/ml$) significantly inhibited lipid accumulation in C2C12 skeletal muscle cells. 7. EF (10 ${\mu}g/ml$) significantly increased the mRNA expression of ACOX, HD, VLCAD and MCAD in NMu2Li liver cells compared with control. Conclusions : These results suggest that EF may prevent obesity by increasing the mRNA expression of mitochondrial fatty acid ${\beta}$-oxidizing enzymes in 3T3-L1 adipocytes, by not only regulating the fatty acid oxidation through activation of AMPK and PPAR${\alpha}$, but also increasing the UCP2 mRNA expression in C2C12 skeletal muscle cells, and by stimulating the mRNA expression of fatty acid-oxidizing enzymes in NMu2Li liver cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6517-6521
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• 2012

To analyze the growth, proliferation, apoptosis, invasiveness and chemotherapy sensitivity of EC9706 cells after K-Ras gene silencing, an expression carrier pSilencer-siK-Ras was constructed, and the EC9706 cell line was transfected using a liposome technique. Six groups were established: Control, siRNA NC (transfected with empty vector pSilencer2.1); Ras siRNA (transfected with pSilencer-siK-Ras2); Paclitaxel; Paclitaxel + siRNA NC; and Ras siRNA + Paclitaxel. After the treatment, RT-PCR, Western blotting, MTT assay, flow cytometry and the Transwell technique were used to assess expression of K-Ras mRNA and protein in EC9706 cells, as well as cell growth, proliferation, apoptosis and invasiveness. The effect of Paclitaxel chemotherapy was also tested. pSilencer-siK-Ras2 effectively down-regulated expression of K-Ras mRNA and protein in EC9706 cells, growth being significantly inhibited. Flow cytometry indicated obvious apoptosis of cells in the experimental group, with arrest in the G1 phase; cell migration ability was also reduced. After pSilencer-siK-Ras2 transfection or the addition of Paclitaxel, EC9706 cells were suppressed to different extents; the suppressive effect was strengthened by combined treatment. The results suggested that RNAi-induced K-Ras gene silencing could enhance chemotherapy sensitivity of esophageal cancer.

• Restorative Dentistry and Endodontics
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• v.31 no.6
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• pp.437-444
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• 2006

This study was aimed to elucidate the biological function of OD314 (Apin protein), which is related to ameloblast differentiation and amelogenesis. Apin protein, calcifying epithelial odontogenic (pindborg) tumors (CEOTs)-associated amyloid, were isolated from CEOTs, and has similar nucleotide sequences to OD314. We examined expression of the OD314 mRNA using in-situ hybridization during tooth development in mice. Expression of OD314 and several enamel matrix proteins were examined in the cultured ameloblast cell line up to 28 days by reverse transcription-polymerase chain reaction (RT-PCR) amplification. After inactivation and over-expression of the OD314 gene in ameloblast cell lines using U6 vectordriven RNA interference and CMV-OD314 construct, RT-PCR were performed to evaluate the effect of the OD314 during amelogenesis. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were more strongly expressed in ameloblast than odontoblast. 2. When ameloblast cells were cultured in the diffcrentiation and mineralization medium for 28 days, the tuftelin mRNA expression was maintained from the beginning to day 14, and then gradually decreased to day 28. The expressions of amelogenin and enamelin were gradually decreased according to the ameloblast differentiation. 3. Inactivation of OD314 by U6-OD314 siRNA construct down-regulated the expression of OD314, MMP-20, and tuftelin, whereas over-expression of OD314 by CMV-OD314 construct up-regulated the expression of OD314 and MMP-20 without change in tuftelin. These results suggest that OD314 is considered as an ameloblast-enriched gene and may play the important roles in ameloblast differentiation and mineralization.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2799-2806
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• 2012

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue ($0.540{\pm}0.083$) was significantly higher than in adjacent normal bladder tissue ($0.492{\pm}0.070$) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.

• The Korea Journal of Herbology
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• v.26 no.1
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• pp.65-74
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• 2011

Objectives : This study was undertaken to verify the modulation mechanism of Gyeongshingangjeehwan18 (GGEx18) in ob/ob male mice. Methods : Eight-week old mice (wild-type C57BL/6J and ob/ob) were used for all experiments. Wild-type C57BL/6J mice were used as lean control and obese ob/ob mice were randomly divided into 5 groups : obese control, GGEx15 (Ephedra sinica Stapf + Rheum palmatum L.), GGEx16 (Ephedra sinica Stapf + Laminaria japonica Aresch), GGEx17 (Rheum palmatum L. + Laminaria japonica Aresch), and GGEx18 (Ephedra sinica Stapf + Laminaria japonica Aresch + Rheum palmatum L.). After mice were treated with several kinds of GGEx for 11 weeks, the mRNA expression of peroxisome proliferator-activated receptor (PPAR) target genes and uncoupling protein (UCP) were measured. In addition, $PPAR{\alpha}$ and $PPAR{\beta}$ transactivation was examined in NMu2Li hepatocytes, C2C12 myocytes, and 3T3-L1 preadipocytes using transient transfection assays. Results : 1. Hepatic $PPAR{\alpha}$ target genes, such as ACOX and VLCAD mRNA levels were significantly increased by GGEx18 compared with obese controls. In skeletal muscle, LCAD mRNA expression was stimulated by GGEx16, GGEx17, and GGEx18, whereas MCAD mRNA expression by GGEx17 and GGEx18. $PPAR{\beta}$ target LPL mRNA levels were also increased by GGEx16, GGEx17, and GGEx18 in skeletal muscle, but adipose LPL mRNA levels were decreased. In addition, GGEx18 upregulated UCP mRNA expression in skeletal muslce. 2. $PPAR{\alpha}$ reporter gene expression was increased by GGEx18 in NMu2Li cells compared with vehicle. $PPAR{\alpha}$ and $PPAR{\beta}$ reporter activities were also increased by all GGEx treatments in C2C12 and 3T3-L1 cells. Conclusions : These results suggest that GGEx can act as $PPAR{\alpha}$ and $PPAR{\beta}$ activators, and that GGEx may regulate obesity by stimulating $PPAR{\alpha}$, $PPAR{\beta}$, and UCP activity. Of the 4 compositions, GGEx18 seems to be most effective in improving obesity and lipid disorders.

• Journal of fish pathology
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• v.37 no.1
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• pp.1-8
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• 2024

The advantages of replicon vectors of RNA viruses include a high ability to stimulate innate immunity and exponential amplification of target mRNA leading to high expression of foreign antigens. The present study aimed to construct a DNA-layered nervous necrosis virus (NNV) replicon vector system in which the capsid protein gene was replaced with a foreign antigen gene and to compare the efficiency of foreign antigen expression between the conventional DNA vaccine vector and the present replicon vector. We presented the first report of a nodavirus DNA replicon-based foreign antigen expression system. Instead of a two-vector system, we devised a one-vector system containing both an NNV RNA-dependent RNA polymerase cassette and a foreign antigen-expressing cassette. This single-vector approach circumvents the issue of low foreign protein expression associated with the low co-transfection efficiency of a two-vector system. Cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 (with the capsid gene ORF replaced with VHSV glycoprotein ORF) exhibited significantly higher transcription of the VHSV glycoprotein gene compared to cells transfected with either a vector without hammerhead ribozyme or a conventional DNA vaccine vector expressing the VHSV glycoprotein. Furthermore, the transcription level of the VHSV glycoprotein in cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 showed a significant increase over time. These results suggest that NNV genome-based DNA replicon vectors have the potential to induce stronger and longer expression of target antigens compared to conventional DNA vaccine vectors.

• Biomedical Science Letters
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• v.15 no.3
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• pp.179-185
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• 2009

In our previous report, we showed that $PPAR{\gamma}$ does not influence adipogenesis in females with functioning ovaries, indicating that $PPAR{\gamma}$ activity on adipogenesis is associated with sex-related factors. Among the sex-related factors, estrogen has been recognized as a major factor in inhibiting adiposgenesis in females. Thus, we hypothensized that $17{\beta}$-estradiol (E) inhibits 3T3-L1 cell adipogenesis by preventing $PPAR{\gamma}$ activity. E decreased triglyceirde accumulation in differentiated 3T3-L1 cells compared with control group. E also decreased the expression of $PPAR{\gamma}$ mRNA as well as $PPAR{\gamma}$ dependent adipocyte-specific genes, such as adipocyte fatty acid binding protein and tumor necrosis factor $\alpha$. In addition, E not only decreased luciferase reporter activity by $PPAR{\gamma}$, but also transfection of estrogen receptor $\alpha$ ($ER{\alpha}$) or $ER{\beta}$ led to decreases in $PPAR{\gamma}$ reporter gene activation. Moreover, E-activated ERs significantly decreased the luciferase reporter gene activation induced by $PPAR{\gamma}$ transfection, suggesting that estrogen-activated ERs inhibit $PPAR{\gamma}$-dependent transactivation. Accordingly, our results demonstrate that E inhibits the action of $PPAR{\gamma}$ on adipogenesis through E activated ER, providing evidence that lack of estrogen may potentiate $PPAR{\gamma}$ action on adipogenesis.

• YAKHAK HOEJI
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• v.52 no.1
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• pp.67-72
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• 2008

The use of doxorubicin, which is one of the most effective anticancer agents, is often limited by occurrence of acquired resistance in tumor cells. GSH has been shown to be involved in the development of this drug resistance. Transcription factor Nrf2 governs the expression of GSH synthesizing glutamylcysteine ligase (GCL), as well as multiple phase 2 detoxifying enzymes. Here we show that Nrf2 is one of factors determining doxorubicin sensitivity. Nrf2-deficient fibroblasts (murine embryonic fibroblasts, MEF) were more susceptible to doxorubicin mediated cell death than wild-type cells. Doxorubicin treatment elevated levels of Nrf2-regulated genes including NAD(P)H: quinone oxidoreductase (Nqo1) and GCL in wild-type fibroblasts, while no induction was observed in Nrf2-deficient cells. Doxorubicin resistance in human ovarian SK-OV cells was reversed by treatment with L-buthionine-sulfoxamine (BSO), which is depleting intracellular GSH. Finally, transfection of SK-OV cells with Nrf2 siRNA resulted in exacerbated cytotoxicity following doxorubicin treatment compared to scrambled RNA control. These results indicate that the Nrf2 pathway, which plays a protective role in normal cells, can be a potential target to control cancer cell resistance to anticancer agents.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1785-1790
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• 2013

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the $NAD^+$-dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.