• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.84-84
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• 2003

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. DNA methylation is a highly plastic and critical component of mammalian development The DNA methyltransferases (Dnmts) are responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. The maintenance DNA methyltransferase enzyme, Dnmt 1, and the de novo methyltransferase, Dnmt3a and Dnmt3b, are indispensable for development because mice homozygous for the targeted disruption of any of these genes are not viable. The occurrence of DNA methylation is not random, and it can result in gene silencing The mechanisms underlying these processes are poorly understood. It is well established that DNA methylation and histone deacetylation operate along a common mechanistic pathway to repress transcription through the action of methyl-binding domain proteins (MBDs), which are components of, or recruit, histone deacetylase (HDAC) complexes to methylated DNA. As a basis for future studies on the role of the DNA-methyl-transferase in porcine development, we have isolated and characterized a partial cDNA coding for the porcine Dnmt1. Total RNA of testis, lung and ovary was isolated with TRlzol according to the manufacture's specifications. 5 ug of total RNA was reverse transcribed with Super Script II in the presence of porcine Dnmt 1 specific primers. Standard PCRs were performed in a total volume of 50 ul with cDNA as template. Two DNA fragmenets in different position were produced about 700bp, 1500bp and were cloned into pCR II-TOPO according to the manufacture's specification. Assembly of all sequences resulted in a cDNA from 158bp of 5'to 4861bp of 3'compare with the known human maintenance methyltransferase. Now, we are cloning the unknown Dnmt 1 region by 5'-RACE method and expression of Dnmt 1 in tissues from adult porcine animals.

• Korean Journal of Environmental Biology
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• v.40 no.3
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• pp.255-266
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• 2022

In crustaceans, molting is regulated by interactions between ecdysteroid and juvenile hormone (JH) signaling pathway-related genes. Unlike the ecdysteroid signaling pathway, little information on the role of JH signaling pathway-related genes in molting is available in zooplanktonic crustaceans. In this study, three genes (juvenile hormone acid O-methyltransferase (JHAMT), methoprene-tolerant (Met), and juvenile hormone epoxide hydrolase (JHEH)) which are involved in the synthesis, receptor-binding, and degradation of JH were identified using sequence and phylogenetic analysis in the brackish water flea, Diaphanosoma celebensis. Transcriptional changes in these genes during the molting cycle in D. celebensis were analyzed. Sequence and phylogenetic analysis revealed that these putative proteins may be functionally conserved along with those of insects and other crustaceans. In addition, the expression of the three genes was correlated with the molting cycle of D. celebensis, indicating that these genes may be involved in the synthesis and degradation of JH, resulting in normal molting. This study will provide information for a better understanding of the role of JH signaling pathway-related genes during the molting process in Cladocera.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.4
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• pp.417-426
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• 2023

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

• Journal of Life Science
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• v.33 no.11
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• pp.876-886
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• 2023

In some cases of head and neck cancers (HNC), surgical interventions may result in the loss of organs and/or changes to their functions, thereby significantly affecting the patient's quality of life. As a result, the surgical treatment of HNC patients is often limited to specific cases, and alternative treatment modalities, such as chemotherapy, are considered. However, serious adverse effects caused by chemotherapy, such as severe nausea and vomiting, necessitate the need for the development of adjunctive methods to minimize patient suffering. Chuanxiong, Ligusticum chuanxiong (L. chuanxiong), is a natural herb used in Eastern medicine to treat cerebrovascular disorders and headaches. This study aimed to predict the effect and potential of L. chuanxiong as an auxiliary anticancer drug through network-based pharmacology and molecular docking analysis. The study results showed that 40 out of 41 genes of L. chuanxiong shared common targets of HNC and their proteins could be used to target HNC cells to prevent cancer progression. The results of the functional enrichment analysis confirmed that L. chuanxiong is associated with the neuroactive-ligand metabolism and neurotransmitter pathways, indicating its potential medicinal value as an adjuvant in HNC treatment. Lastly, our findings demonstrated that the active ingredient of L. chuanxiong, (Z)-Ligustilide, has the ATP binding site of heat shock protein 90, a protein known to promote the activation of cancer cells. These results suggest that L. chuanxiong is a promising candidate for developing auxiliary anticancer drugs, and further research could potentially lead to the discovery of newer and safer anti-cancer agents.

• IMMUNE NETWORK
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• v.24 no.2
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• pp.3.1-3.23
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• 2024

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68⁺ cell number and the levels of iNOS, TNF-α, IL-1β (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO^-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

• IMMUNE NETWORK
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• v.23 no.4
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• pp.33.1-33.13
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• 2023

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been acknowledged as an effective mean of preventing infection and hospitalization. However, the emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) has led to substantial increase in infections among children and adolescents. Vaccine-induced immunity and longevity have not been well defined in this population. Therefore, we aimed to analyze humoral and cellular immune responses against ancestral and SARS-CoV-2 variants after two shots of the BNT162b2 vaccine in healthy adolescents. Although vaccination induced a robust increase of spike-specific binding Abs and neutralizing Abs against the ancestral and SARS-CoV-2 variants, the neutralizing activity against the Omicron variant was significantly low. On the contrary, vaccine-induced memory CD4⁺ T cells exhibited substantial responses against both ancestral and Omicron spike proteins. Notably, CD4⁺ T cell responses against both ancestral and Omicron strains were preserved at 3 months after two shots of the BNT162b2 vaccine without waning. Polyfunctionality of vaccine-induced memory T cells was also preserved in response to Omicron spike protein. The present findings characterize the protective immunity of vaccination for adolescents in the era of continuous emergence of variants/subvariants.

• Journal of Nutrition and Health
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• v.57 no.2
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• pp.196-210
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• 2024

Purpose: This study investigated the anti-obesity effects of a combination of Syzygium aromaticum L. and Sorbus commixta Hedl. (SS) in vitro and in vivo. Methods: The extracts of Syzygium aromaticum extract (SA) and Sorbus commixta extract (SC) were prepared individually using distilled water. They were mixed in a 1:2 ratio for use in the experiment. To assess the anti-obesity potential of SS in vitro, we examined cell proliferation, cellular triglyceride (TG), and total cholesterol (TC) levels, as well as lipogenesis and β-oxidation in 3T3-L1 cells. To confirm its anti-obesity potential in vivo, C57BL/6J mice were fed a 60% high-fat diet (HFD) to induce obesity. SA alone, SC alone, and their combination compound, SS (at a dosage of 200 mg/kg) were orally administered for 6 weeks. Thereafter, to conduct a comparative evaluation, serum analysis, western blotting of liver tissues, and histopathological analysis were performed. Results: Both SS200 and SS400 significantly inhibited the cellular TG and TC contents in the 3T3-L1 cells. Furthermore, treatment of the cells with SS (at a dose 200 and 400 ㎍/mL) also led to a noticeable regulation of key lipogenic and β-oxidation factors. Treatment of obese mice with SS resulted in a greater reduction in serum leptin and TG levels compared to treatment with the individual compounds (SA and SC). Furthermore, activation of AMP-activated protein kinase α by SS treatment resulted in the suppression of sterol regulatory element-binding proteins (SREBP)-1, leading to the inhibition of acetyl-CoA carboxylase (ACC) expression. Conclusion: Our results suggest that SS may have the potential to prevent obesity through a reduction in the TG and TC levels and regulation of lipogenesis and β-oxidation.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.5
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• pp.651-663
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• 2016

Corn silk, Job's tears, Lentinus edodes, and apple peel 70% ethanol extracts (CS, JT, LE, and AP) were studied for their antioxidant activities. CS among all extracts showed the highest antioxidant activities based on total polyphenol and flavonoid contents, 2,2-diphenyl-${\beta}$-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical scavenging activity, and reducing power. Adipocyte differentiation was investigated by Oil Red O staining assay using CS, JT, LE, AP, and extract of developed bread containing corn silk, Job's tears, Lentinus edodes, and apple peel (DB) treated to 3T3-L1 adipocytes. DB1 and DB2 showed anti-adipogenic and antioxidant effects. Triglyceride (TG) accumulation in 3T3-L1 cells was measured, and among the samples tested (CS, JT, LE, and AP), CS was found to have the highest inhibitory activity against TG accumulation of differentiated 3T3-L1 adipocytes and regulated factors associated with adipogenesis. CS suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. We examined the effects of CS on the levels of CCAAT-enhancer-binding protein ${\beta}(C/EBP{\beta})$, peroxisome proliferator activated receptor ${\gamma}(PPAR{\gamma})$, and adipocyte-specific lipid binding protein (aP2) mRNA as well as protein levels in 3T3-L1 cells treated with CS at various concentrations (0, 10, 50, and $100{\mu}g/mL$) during adipocyte differentiation and treatment with CS in 3T3-L1 adipocytes down-regulated expression of $PPAR{\gamma}$ and aP2 mRNA. CS also significantly inhibited up-regulation of $C/EBP{\beta}$, $PPAR{\gamma}$, and aP2 proteins during adipocyte differentiation. These data indicate that DBs have anti-adipogenic activity induced by CS in 3T3-L1 preadipocytes, and CS exerts anti-adipogenic activity by inhibiting expression of $C/EBP{\beta}$, $PPAR{\gamma}$, and aP2 signaling pathway in 3T3-L1 adipocytes. JT, LE, and AP had no inhibitory effects on differentiation of 3T3-L1 preadipocytes but displayed strong antioxidant effects. These results suggest that the developed bread may be a health beneficial food that can prevent or treat obesity and diseases induced by oxidative stress.

• Journal of Nutrition and Health
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• v.48 no.1
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• pp.9-18
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• 2015

Purpose: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. Methods: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein ($C/EBP{\beta}$). Results: The total polyphenol and flavonoid content of PF-W was $52.15{\pm}4.02$ and $6.56{\pm}0.47mg/g$, respectively. PF-W treatment decreased LPL content in media to $58{\pm}5%$ of that in control adipocyte media, and increased LPL content to $117{\pm}3.5%$ of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of $C/EBP{\beta}$, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. Conclusion: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through $C/EBP{\beta}$-mediated induction of SorLA expression.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.561-574
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• 2000

Poly-P has been used to prevent decomposition of foods and has been shown to have inhibitory effect on the growth of gram positive bacteria. The purpose of this study was to evaluate the effect of poly-P on the growth of Porphyromonas endodontalis, a gram negative obligate anaerobic rod, endodontopathic bacterium. P. endodontalis ATCC 35406 was in BHI broth containing hemin and vitamin K with or without poly-P. Inhibitory effect of each poly-P which was added at the beginning(lag phase) or during(exponential phase) the culture, MIC(minimum inhibitory concentration) was determined by measuring the optical density of the bacterial cell at 540nm. Viable cell counts were measured to determined whether poly-P has a bactericidal effect. Leakage of intracellular nucleotides from P. endodontalis was determined at 260nm and morphological change of P. endodontalis was observed under the TEM(transmission electron microscope). Binding of 32P-labeled poly-P to P. endodontalis was examined. SDS-polyacrylamide gel electrophoresis and zymography were performed to observe the changes in protein and enzyme profiles of P. endodontalis, respectively. The results from this study were as follows : 1. The minimal inhibitory concentration(MIC) of poly-P to P. endodontalis appeared to be 0.04~0.05%. 2. Poly-P added to the P. endodontalis culture during the exponential phase of P. endodontalis was as much effective as poly-P added at the begining of the culture, suggesting that the antibacterial effect of poly-P is not much dependent on the initial inoculum size of P. endodontalis. 3. Poly-P are bactericidal to P. endodontalis, demonstrating the decrease of the viable cell counts. 4. Intracellular nucleotide release from the P. endodontalis, was not increased in the presence of poly-P and was not reversed by the addition of divalent cations like $Ca^{2+}$ and $Mg^{2-}$. 5. Under the TEM, it was observed that fine electro-dense materials were prominent in the poly-P grown P. endodontalis, appearing locally in the cell, and the materials were more abundant and more dispersed in the cell as the incubation time with poly-P increased. In addition, highly electron dense granules accumulated in many poly-P grown cells, most of which were atypical in their shape. 6. Binding of 32P-labeled poly-P to P. endodontalis appeared to be 32.8 and 45.5 and 53.4% at 30 minutes, 1 hours and 2 hours, respectively. 7. In the presence of poly-P. the synthesis of proteins with apparent molecular masses of 25, 27, 35, 45 was lost or drastically decreased whereas expression of a protein with an apparent molecular mass of 75 was elevated. 8. Proteolytic activity of P. endodontalis was decreased by poly-P. The overall results suggest that use of poly-P may affect the growth of P. endodontalis, and the anti-bacterial activity of poly-P seems largely bactericidal. Changes in shape, protein expression, and proteolytic activity of P. endodontalis by poly-P may be directly and indirectly attributed to the antibacterial effect of poly-P. Further studies will be needed to confirm the effect of poly-P.