• Journal of Periodontal and Implant Science
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• v.36 no.3
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• pp.767-782
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• 2006

이 연구의 목적은 DNA microarray 분석법을 이용하여 건강한 사람치주인대섬유모세포, 건강한 사람치은섬유모세포, 복제노화된 사람치은섬유모세포, 염증성 사람치주인대 섬유모 세포의 유전자 발현 형태를 상호비교하고자 하였다. 환자의 동의하에 충치, 치주염이 없이 교정발치된 치아의 치주인대세포를 배양하여 건강한 치주인대섬유모세포로, 만성치주염으로 발거된 치아에서 채취하여 배양한 세포를 염증성 치주인대섬유모세포로 선정하였다. 구강에서 채취한 치은결체조직에서 배양한 사람치은섬유모세포를 일차 배양한 후 계대배양을 통해 복제 노화를 유도하였다. $-198^{\circ}C$의 액화질소에 저장되어 있던 2, 4, 8, 15, 16세대 세포를 실험에 이용하였다. 위의 모든 세포들은 60 mm 배양접시에서 세포들이 80-90%의 밀생이 될 때까지 5% $CO_2$, $37^{\circ}C$, 100% 습도의 배양기에서 2일 간격으로 10% FBS가 함유된 DMEM 세포 배양액을 교체하면서 세포를 배양하였다. Trizol Reagent (Invitrogen, USA)를 이용하여 제조회사의 지시에 따라 total RNA를 추출하였다. 18S RNA와 28S RNA를 확인한 후 DNA microarray 분석을 실시하였다. 4배수 이상의 변화양상을 비교시 상호 유전자 발현의 차이를 나타내었다. 건강한 사람치은섬유모세포(2세대)와 노화된 치은섬유모세포에서 가장 높은 발현변화를 나타낸 반면 DMC1 dosage suppressor of mck1 homolog, meiosis-specific homologous recombination,은 건강한 치은섬유모세포에서 가장 높게 나타났다. 염증성 치은인대섬유모세포와 건강한 치주인대 섬유모세포를 비교시, Regucalcin은 염증성 치주인대섬유모세포에서 가장 높게 나타났고, Vascular cell adhesion molecule 1도 두 번째로 높게 나타났다. 건강한 치주인대섬유모 세포와 건강한 치은섬유모세포를 비교시, IL-11과 periostin이 치주인대섬유모세포에서 높은 발현을 나타낸 반면, Prostaglandin D2 synthase 21kDa과 Thioredoxin interacting protein은 치은섬유모세포에서 높은 발현을 나타내었다. 염증성 치주인대섬유모세포와 노화된 치은섬유모세포(15세대 이상)를 비교시 149개의 유전자가 유사한 발현 수준을 나타내었다. 이 연구는 노화, 염증, 세포 형태에 따라서 유전자 수준에서 가장 높거나 높은 수준 변화를 보이는 유전자가 다를 수 있음을 나타낸다. 향후, 치주염 환자들에서, 노염, 염증, 세포 특이성에 관한 유전자 표시지를 이용하여 진단하거나 치료에 응용하기 위해서는 더 많은 연구가 필요하리라 사료된다.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.115-122
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• 1998

The maintenance of a viable pregnancy has long been viewed as an immunological paradox. The deveolping embryo and trophoblast are immunologically foreign to the maternal immune system due to their maternally inherited genes products and tissue-specific differentiation antigens (Hill & Anderson, 1988). Therefore, speculation has arisen that spontaneous abortion may be caused by impaired maternal immune tolerance to the semiallogenic conceptus (Hill, 1990). Loss of recall antigen has been reported in immunosuppressed transplant recipients and is associated with graft survival (Muluk et al., 1991; Schulik et al., 1994). Progesterone $(10^{-5}M)$ has immunosuppressive capabilities (Szekeres-Bartho et al., 1985). Previous study showed that fertile women, but not women with unexplained recurrent abortion (URA), lose their immune response to recall antigens when pregnant (Bermas & Hill, 1997). Therefore, we hypothesized that immunosuppressive doses of progesterone may affect proliferative response of lymphocytes to trophoblast antigen and alloantigen. Proliferative responses using $^3H$-thymidine ($^3H$-TdR) incorporation of peripheral blood mononuclear cells (PBMCs) to the irradiated allogeneic periperal blood mononuclear cells as alloantigen, trophoblast extract and Flu as recall antigen, and PHA as mitogen were serially checked in 9 women who had experienced unexplained recurrent miscarriage. Progesterone vaginal suppositories (100mg b.i.d; Utrogestan, Organon) beginning 3 days after ovulation were given to 9 women with unexplained RSA who had prior evidence of Th1 immunity to trophoblast. We checked proliferation responses to conception cycle before and after progesterone supplementation once a week through the first 7 weeks of pregnancy. All patients of alloantigen and PHA had a positive proliferation response that occmed in the baseline phase. But 4 out of 9 patients (44.4%) of trophoblast antigen and Flu antigen had a positive proliferative response. The suppression of proliferation response to each antigen were started after proliferative phase and during pregnancy cycles. Our data demonstrated that since in vivo progesterone treated PBMCs suppressed more T-lymphocyte activation and $^3H$-TdR incorporation compare to PBMCs, which are not influenced by progesterone. This data suggested that it might be influenced by immunosuppressive effect of progesterone. In conclusion, progesterone may play an important immunological role in regulating local immune response in the fetal-placental unit. Furthermore, in the 9 women given progesterone during a conception cycle, Only two (22%) repeat pregnancy losses occured in these 9 women despite loss of antigen responsiveness (one chemical pregnancy loss and one loss at 8 weeks of growth which was karyotyped as a Trisomy 4). These finding suggested that pregnancy loss due to fetal aneuploidy is not associated with immunological phenomena.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.2
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• pp.82-89
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• 2016

Objective: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. Methods: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. Results: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (${\pm}2.66$) vs. 75.40 (${\pm}4.92$); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (${\pm}4.79$) vs. 67.79 (${\pm}5.17$); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. Conclusion: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.3
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• pp.181-191
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• 2008

Objective: Environmental chemicals alter reproduction, growth, and survival by changing the normal function of the endocrine system. Bisphenol A (BPA), one of the endocrine disruptors, is known to be an estrogen receptor agonist. Therefore, we hypothesized that BPA may affect male reproduction including spermatogenesis in the mouse testis. Methods: We used 7-week-old ICR mice. The first experiment group received BPA in sesame oil (vehicle, 1 mg/kg, 10 mg/kg, and 100 mg/kg) by i.p. injection and mice were sacrificed 24 hr later. The second experiment group received BPA (vehicle, 10 ${\mu}g/kg$, 1 mg/kg, and 100 mg/kg) daily for 14 days by subcutaneous injection. Expression of cell type-specific marker genes in the testis was evaluated by RT-PCR. Histological analysis, immunofluorescence staining, and TUNEL staining were also performed. Results: RT-PCR analyses showed that expression of luteinizing hormone receptor (LHR), a marker gene for the Leydig cell, was notably decreased in the testes of high dose-exposed mice. No obvious difference in the histology of testes was noted among treatment groups. Immunostaining of LHR in the first experiment group did not show noticeable difference in LHR protein expression in Leydig cells. Immunohistochemistry also revealed heightened expression of the immunoreactive Bax in the treatment group, and this was accompanied by positive TUNEL staining in the interstitial area within testis where Leydig cells reside. Conclusions: Our result suggests that BPA affects Leydig cell functions by altering gene expression and by increasing apoptosis in the mouse testis.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.37-37
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• 2015

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed an identical sequence to known RdRp genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that, although variations in the growth rate existed among progeny and virus infection was observed in highly actively growing progeny, there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny. This study attempted to cure the edible mushroom L. edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygen-depleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no $discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. We were also explored effects of LeV on fruiting body formation and mushroom yield. The fruiting body formation yield of virus-free L. edodes was larger than virus-infected L. edodes. These results indicate that LeV infection has a deleterious effect on mycelial growth and fruiting body formation. In addition, we have been investigated host-parasite interaction between L. edodes and its mycovirus interaction to study viral mechanism by establishment of proteomics.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.213-218
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• 2010

Lactate dehydrogenases (LDHs) have been highly focused for long time, due to their important roles in biochemical and metabolic pathways of cells. On the basis of genome-wide searching results, three putative LDH genes from Bacillus cereus ATCC 14579 genome have been PCR-amplified, cloned, and well-expressed in E. coli. All three BcLDH isozymes are supposed to share highly conserved catalytic amino acid residues in common $NAD^+$-dependent LDHs. Meanwhile, BcLDH1 consisting of 314 amino acids shares 86 and 49% of identities with BcLDH2 and 3, respectively. Interestingly, only BcLDH1 showed the converting activities between L-lactate and pyruvate in the presence of $NAD^+$ coenzyme, while the other isozymes are likely to have almost no activity. As a result, it was revealed that BcLDH1 can be a typical $NAD^+$-dependent L-lactate-specific dehydrogenase.

• Journal of Life Science
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• v.25 no.3
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• pp.285-292
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• 2015

Citrus peel (CP) is used as a traditional herb with diverse beneficial pharmacological activities, such as anti-inflammatory, anti-oxidant, and anti-allergic effects. However, the anti-obesity effects of citrus peel are poorly defined. The aim of this study was to evaluate ethanol extracts of citrus peel (EECP) for its adipocyte differentiation and adipogenesis in 3T3-L1 preadipocytes. The aim of this study was to evaluate an EECP for its adipocyte differentiation and adipogenesis in 3T3-L1 preadipocytes. Treatment with EECP significantly suppressed the terminal differentiation of 3T3-L1 preadipocytes in a dose-dependent manner, as confirmed by a decrease in lipid droplet number and lipid content and an accumulation of cellular triglyceride. EECP exhibited potential adipogenesis inhibition and downregulated the expression of pro-adipogenic transcription factors, such as sterol regulatory elementbinding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancerbinding proteins α (C/EBPα) and C/EBPβ, and adipocyte expressed genes, such as adipocyte fatty acid binding protein (aP2) and Leptin. In addition, EECP treatment effectively activated the AMP-activated protein kinase (AMPK) signaling pathway; however, compound C, a specific inhibitor of AMPK, significantly reduced the EECP-induced inhibition of adipogenesis. Taken together, these results indicate EECP showed strong anti-obesity effects through the AMPK signaling pathway, and further studies will be needed to identify the active compounds that confer the anti-obesity activity of EECP.

• Journal of Life Science
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• v.19 no.8
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• pp.1164-1169
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• 2009

Aberrant DNA methylation plays an important role in the development of cancer. It has been reported recently that DNA hypermethylation is involved in the maintenance of cancer stem cells. The present study was designed to test the hypothesis that the demethylating agent, 5-aza-2'-deoxycytidine (AZA), can inhibit the potential for maintenance of cancer stem cells. To validate this hypothesis, we used 4T1 syngeneic mouse models of breast cancer. The AZA pre-treated 4T1 cells showed a dramatic inhibition of tumorsphere formation, compared to their counterparts in vitro. In addition, the AZA treatment significantly suppressed the expression of stem regulator genes, such as oct-4, nanog and sox2, compared to counterparts in vivo. Therefore, selective inhibition of DNA methylation may be useful for stem-specific cancer therapy.

• Journal of Life Science
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• v.18 no.3
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• pp.336-343
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• 2008

Histone deacetylases (HDACs) inhibitors have emerged as the accessory therapeutic agents for various human cancers, since they can block the activity of specific HDACs, restore the expression of some tumor suppressor genes and induce cell differentiation, cell cycle arrest and apoptosis in vitro and in vivo. In the present study, we investigated that the effect of trichostatin A (TSA), an HDAC inhibitor, on the cell growth and apoptosis, and its effect on the nuclear factor-kappaB $(NF-{\kappa}B)$ activity in 267B1 human prostate epithelial cells. Exposure of 267B1 cells to TSA resulted in growth inhibition and apoptosis induction in and dose-dependent manners as measured by fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. TSA treatment inhibited the levels of IAP family members such as c-IAP-1 and c-IAP-2 and induced the proteolytic activation of caspase-3, -8 and -9, which were associated with concomitant degradation of poly (ADP-ribose)-polymerase, ${\beta}-catenin$ and laminin B proteins. The increase in apoptosis by TSA was connected with the translocation of $NF-{\kappa}B$ from cytosol to nucleus, increase of the DNA binding as well as promoter activity of $NF-{\kappa}B$, and degradation of cytosolic inhibitor of KappaB $(I{\kappa}B)-{\alpha}$ protein. We therefore concluded that TSA demonstrated anti-proliferative and apoptosis-inducing effects on 267B1 cells in vitro, and that the activation of caspases and $NF-{\kappa}B$ may play important roles in its mechanism of action. Although further studies are needed, these findings provided important insights into the possible molecular mechanisms of the anti-cancer activity of TSA.

• Journal of Life Science
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• v.23 no.10
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• pp.1216-1222
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• 2013

NK cells are lymphoid immune cells that participate in innate immunity to protect against foreign pathogens and transforming cells. It is known that the activity of NK cells is regulated by a balance between activating and inhibitory signals rather than specific antigens. One important activating signal is mediated by the NKG2D receptor, which recognizes NKG2D ligands on cancer cells. Therefore, tumor cells that express sufficient amounts of NKG2D ligands could be eliminated by NKD2D+ cells, including NK cells. Oncogenes drive tumor cells to apoptosis resistant and uncontrolled proliferation by altered expression of many critical genes. Therefore, the expression of NKG2D ligands may be affected by oncogenes. This study focused on increasing the susceptibility of cancer cells to NK cells by regulating the expression of NKG2D ligands influenced by three oncogenes: k-ras, wnt, and c-myc. We demonstrated that inhibition of k-ras and c-myc increased the expression of NKG2D ligands and enhanced the susceptibility of cancer cells to NK cells. On the contrary, inhibition of the wnt pathway decreased MICA and ULBP1 transcripts. Although the decreased transcription of NKG2D ligands by inhibition of the wnt pathway, surface proteins of NKG2D ligands were not changed, and the susceptibility of HCT-116 cells was unaffected. The results demonstrate that the transcription of NKG2D ligands are regulated deferentially by the k-ras, c-myc, and wnt pathways and that the cytotoxicity of NK cells solely depends on the amount of surface NKG2D ligands.