• 한국발생생물학회지:발생과생식
• /
• 제11권3호
• /
• pp.195-203
• /
• 2007

자웅이체형 어류의 성결정 및 성분화는 일반적으로 각 개체의 유전형을 따른다. 자연환경에서는 자신이 가진 유전정보의 조절에 따라 성분화 시기에 아로마테이즈 유전자의 발현이 증가하거나 감소하고, 그 결과 스테로이드 호르몬들의 조성이 결정되어 각기 다른 방향으로 성분화하는 것으로 알려져 있다. 본 연구에서는 해산 태생 어류인 조피볼락(Sebasts schlegeli)의 ovarian type aromatase (P450aromA)와 brain type aromatase (P450aromB)의 유전자를 부분적으로 클로닝하여, 이들의 염기서열을 밝혔으며, 각 유전자에 대한 primer를 제작한 후 성분화 기간 중 이 유전자들의 발현을 조사하였다. 조피볼락 아로마테이즈 유전자들은 조사를 시작한 출산 후 35일째에 여러 개체의 머리와 몸에서 각각 발현되었으나, 52일째에는 아로마테이즈 유전자들의 발현 개체수가 현저히 감소하였다. 그리고 출산 59일째에는 발현 개체 수가 다시 증가하였다. P450aromA과 P450aromB의 발현 양상은 전반적으로 유사하였으나, 45일째에는 P450aromB의 발현 개체수가 P450aromA 발현 개체 수보다 훨씬 많았다. 조직학적 분석을 통해 제시된 이 종의 성분화 시기는 지금까지 출산 $50{\sim}65$일 전후로 알려져 왔으나, 본 연구에서 나타난 아로마테이즈 유전자들의 발현 결과를 볼 때, 이 종의 실질적인 성분화는 알려진 것보다 최소한 $1{\sim}2$주 정도 빨리 진행될 가능성이 있다. 본 연구의 결과는 또한 태생어류의 성분화도 난생어류의 성분화에서 보고된 것과 마찬가지로 아로마테이즈의 작용과 밀접하게 연관되어 있다는 사실을 시사한다.

• 한국발생생물학회지:발생과생식
• /
• 제13권3호
• /
• pp.173-181
• /
• 2009

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (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. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

• Korean Journal of Acupuncture
• /
• 제25권3호
• /
• pp.147-166
• /
• 2008

Objectives : Crohn's disease (CD) is characterized by a chronic relapsing inflammation of the bowel in which proinflammatory cytokines play an important perpetuating role. Methods : Mice (preventive animal model of gliotoxin) were treated with 5 % 2,4,6-trinitrobenzenesulfonic acid (TNBS) at day 1 and day 7. To investigate preventive effects of acupuncture with Gujin at $LI_{11}$, acupuncture was carried out at day -1, day 1, day 3. And, to investigate therapeutic effects, acupuncture with Gujin was carried out at day 3, day 5, day 7. For the data analysis, we checked weight and width of colon, diarrhea, edema, survival rate, changes of body weight, and myeloperoxygenase (MPO) activity. For analysing protein expression, we carried out immunohistochemical staining and Western blot and we analyzed mRNA expression by RT-PCR. Results : Colon of TNBS treated mice was erosive and shortening compared with the colon of control mice and induced damages of colon epithelial cell layer and induced infiltration of immune cells in all layer of colon. Acupuncture of gujin at $LI_{11}$ in preventive mode suppressed macorscopic damages such as erosive and shortening of colon by TNBS and damages of intestinal epithelial cells and infiltration of immune cells in the colon. The average weight of 5 cm distal colon was increased in TNBS treated mice (758${\mu}g$) compared with in control mice (112${\mu}g$) and width of distal colon was also increased in TNBS treated mice (4.9mm) compared with in control mice (1.3mm). Acupuncture with Gujin at $LI_{11}$ in preventive and therapeutic mode suppressed increase of colon weight and width by TNBS. TNBS induced edema of colon and diarrhea and Acupunctured with Gujin at $LI_{11}$ in preventive and therapeutic mode ameliorated these symptom by TNBS. In preventive and therapeutic mode, the effects of acupuncture with Gujin at $LI_{11}$ were increasing the motility, suppressing body weight decreasing, suppressing MPO activity, reducing expressing of TNF-${\alpha}$, IL-1b, and ICAM-1 in colon compared with that by TNBS Conclusions : This study demonstrates that acupuncture with Gujin at $LI_{11}$ represents a potential therapeutic method of Crohn's disease.

• 생명과학회지
• /
• 제26권1호
• /
• pp.117-122
• /
• 2016

본 연구에서는 고구마 소주로부터 획득한 에탄올 추출물 및 순차적 유기용매 분획물을 제조하고, 이들 시료가 LPS로 활성화된 마우스 대식세포 RAW 264.7 세포주에서 nitric oxide (NO) 생산과 세포생존율에 미치는 영향을 연구하였다. 처리한 시료 중 ethyl acetate 분획물(KSD-E8-3) 처리군에서 LPS로 염증 유도된 RAW 264.7 세포주의 생존율에는 영향 없이 NO 생성을 현저하게 감소 시킴을 확인하였다. 또한 분획물 KSD-E8-3 처리에 의해서 염증매개 인자인 COX-2, iNOS 그리고 TNF-alpha의 유전자의 발현이 감소되었으며, iNOS 단백질의 발현이 현저히 감소됨을 Western blot으로 확인하였다. 또한, 분획물 KSD-E8-3 처리에 의해 세포내 대표적인 신호 전달 단백질인 3가지의 MAPK 단백질(p38, JNK, ERK1/2)의 인산화가 모두 억제됨을 확인하였다. 종합적으로, 이러한 연구결과는 고구마 소주 주박에 함유된 생리활성 물질에 의한 항염증 활성 및 작용 기전을 이해하는데 도움을 줄 것으로 기대된다.

• 대한한의학방제학회지
• /
• 제19권1호
• /
• pp.195-205
• /
• 2011

Objectives : Ecklonia cava is brown alga(Laminariaceae) which grows is sea, it has antioxidant, diarrhea and anticoagulant effect. In this study, the effect of ethanol extract of Ecklonia cava (EC) on peptidoglycan(PGN) -induced NO production in RAW 264.7 cells was investigated. Methods : In the present study, IL-6 production was measured using the enzyme-linked immunosorbent assay(ELISA), prostaglandin $\E_2$($\PGE_2$) production was measured using the EIA kit, and inducible NO synthase(iNOS), cyclooxygenase-2(COX-2), and mitogen-activated protein kinase(MAPK) activation, as determined by western blot analysis and reverse transcription -polymerase chain reaction(RT-PCR). Results : EC inhibited PGN-induced NO and IL-6 production. Consistent with these observations, the protein expression of iNOS and COX-2 were inhibited by EC. Moreover, EC suppressed the phosphorylation of extracellular signal-regulated kinase(ERK) 1/2 in PGN-induced RAW 264.7. Conclusions : These results suggest that EC has inhibitory effects on PGN-induced $\PGE_2$, NO, and IL-6 production, as well as the expressions of iNOS and COX-2 in the murine macrophage. These inhibitory effects occur through blockades on the MAPKs phosphorylation.

• Journal of Periodontal and Implant Science
• /
• 제36권3호
• /
• pp.783-796
• /
• 2006

The periodontium is a topographically complex organ consisting of epithelial tissue, soft and mineralized tissues. Structures comprising the periodontium include the gingiva, periodontal ligament (PDL) , cementum and the alveolar bone. The molecular mechanism of differentiation in PDL fibroblast cells remain unclear. Amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In this study, the expression pattern of amino acid transport system L was, therefore, investigated in the differentiation of PDL fibroblast cells. To determine the expression level of amino acid transport system L participating in intracellular transport of amino acids in the differentiation of PDL fibroblast cells, it was examined by RT-PCR, observation of cell morphology, Alizaline red-S staining and uptake analysis after inducing experimental differentiation in PDL fibroblast cells isolated from mouse molar teeth. The results are as follows. 1. The LAT1 mRNA was expressed in the early stage of PDL fibroblast cell differentiation. This expression level was gradually reduced by differentiation- inducing time and it was not observed after the late stage. 2. The expression level of LAT2 mRNA was increased in time-dependent manner during differentiation induction of PDL fibroblast cells. 3. There was no changes in. the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during differentiation of PDL fibroblast cells. 4. The expression level of ALP mRNA was gradually increased and the expression level of Col I mRNA was decreased during differentiation of PDL fibroblast cells. 5. The L-leucine transport was reduced by time from the early stage to the late stage in PDL fibroblast cell differentiation. As the results, it is considered that among neutral ammo acid transport system L in differentiation of PDL fibroblast cells, the LATl has a key role in cell proliferation in the early stage of cell differentiation and the LAT2 has an important role in the late stage of cell differentiation for providing cells with neutral amino acids including several essential amino acids.

• 한국식물학회:학술대회논문집
• /
• 한국식물학회 2002년도 춘계학술발표대회:발표눈문요지록
• /
• pp.62-72
• /
• 2002

This study has investigated the biosynthesis and function of the heavy metal binding peptides, the phytochelatins, in plants. PCs are synthesised enzymatically from glutathione by the enzyme PC synthase in the presence of heavy metal ions. Using Arabidopsis thaliana as a model organism cadmium-sensitive, phytochelatin-deficient mutants have been isolated and characterised in previous studies. The cadl mutants have wildtype levels of glutathione, are PC deficient and lack PC synthase activity. Thus, the CADl gene has been proposed to encode PC synthase. The CADl gene was isolated by a positional cloning strategy The gene was mapped and a candidate identified. Each of four cadl mutants had a single base pair change in the candidate gene and the cadmium-sensitive, cadl phenotype was complemented by the candidate gene. This demonstrated the CADl gene had been cloned. A homologous gene in the fission yeast, Schizosaccharomyces pombe was identified through database searches. A targeted-deletion mutation of this gene was constructed and the mutant, like cadl mutants of Arabidopsis, was cadmium-sensitive and PC-deficient. A comparison of the redicted amino acid sequences reveals a highly conserved N-terminal region Presumed to be the catalytic domain and a variable C-terminal region containing multiple Cys residues proposed to be involved in activation of the enzyme by metal ions. Similar genes were also identified in animal species. The Arabidopsis CADl/AtPCSl and S. pombe SpbPCS genes were expressed in E. coli and were shown to be sufficient for glutathione-dependent, heavy metal activate PC synthesis in vitro, thus demonstrating these genes encode PC synthase enzymes. Using RT-PCR, AtPCSl expression appeared to be independent of Cd exposure. However, at higher levels of Cd exposure a AtPCSl-CUS reporter gene construct appeared to be more highly expressed. Using the reporter gene construct, AtPCSl was expressed most tissues. Expression appeared to be greater in younger tissues and same higher levels of expression was observed in some regions, including carpels and the base of siliques. AtPCS2 was a functional gene encoding an active PC synthase. However, its Pattern of expression and the phenotype of a mutant (or antisense line) have not been determined. Assuming the gene is functional then it has clearly been maintained through evolution and must provide some selective advantage. This implies that, at least in some cells or tissue, it is likely to be the dominant PC synthase expressed. This remains to be determined

• Molecules and Cells
• /
• 제41권9호
• /
• pp.868-880
• /
• 2018

Pancreatic cancer (PC) is one of the most aggressive cancers presenting with high rates of invasion and metastasis, and unfavorable prognoses. The current study aims to investigate whether EZH2/miR-139-5p axis affects epithelial-mesenchymal transition (EMT) and lymph node metastasis (LNM) in PC, and the mechanism how EZH2 regulates miR-139-5p. Human PC and adjacent normal tissues were collected to determine expression of EZH2 and miR-139-5p, and their relationship with clinicopathological features of PC. Human PC cell line was selected, and treated with miR-139-5p mimics/inhibitors, EZH2 vector or shEZH2 in order to validate the regulation of EZH2-mediated miR-139-5p in PC cells. Dual-luciferase report gene assay and chromatin immunoprecipitation assay were employed to identify the relationship between miR-139-5p and EZH2. RT-qPCR and Western blot analysis were conducted to determine the expression of miR-139-5p, EZH2 and EMT-related markers and ZEB1/2. Tumor formation ability and in vitro cell activity were also analyzed. Highly-expressed EZH2 and poorly-expressed miR-139-5p were detected in PC tissues, and miR-139-5p and EZH2 expressions were associated with patients at Stage III/IV, with LNM and highly-differentiated tumors. EZH2 suppressed the expression of miR-139-5p through up-regulating Histone 3 Lysine 27 Trimethylation (H3K27me3). EMT, cell proliferation, migration and invasion were impeded, and tumor formation and LNM were reduced in PC cells transfected with miR-139-5p mimics and shEZH2. MiR-139-5p transcription is inhibited by EZH2 through up-regulating H3K27me3, thereby down-regulation of EZH2 and up-regulation of miR-139-5p impede EMT and LNM in PC. In addition, the EZH2/miR-139-5p axis presents as a promising therapeutic strategy for the treatment of PC.

• 대한한방소아과학회지
• /
• 제28권2호
• /
• pp.56-71
• /
• 2014

Objectives In this study, the author tried to investigate whether piryongbang-gamgil-tang (PGGT) significantly affect in vitro airway mucin secretion, PMA- or EGF- or TNF-${\alpha}$-induced MUC5AC mucin production / gene expression from human airway epithelial cells and increase in airway epithelial mucosubstances and hyperplasia of tracheal goblet cells of rats. Materials and Methods For in vitro experiment, confluent RTSE cells were chased for 30 min in the presence of PGGT to assess the effect of PGGT on mucin secretion by enzyme-linked immunosorbent assay (ELISA). Also, effect of PGGT on PMA- or EGFor TNF-${\alpha}$-induced MUC5AC mucin production and gene expression from human airway epithelial cells (NCI-H292) were investigated. Confluent NCI-H292 cells were pretreated for 30 min in the presence of PGGT and treated with PMA (10 ng/ml) or EGF (25 ng/ml) or TNF-${\alpha}$ (0.2 nM) for 24 hrs, to assess both effect of PGGT on PMA- or EGF- or TNF-${\alpha}$-induced MUC5AC mucin production by ELISA and gene expression by reverse transcription-polymerase chain reaction (RT-PCR). For in vivo experiment, the author induced hypersecretion of airway mucus and goblet cell hyperplasia by exposure of rats to $SO_2$ during 3 weeks. Effect of orally-administered PGGT during 2 weeks on increase in airway epithelial mucosubstances from tracheal goblet cells of rats and hyperplasia of goblet cells were assesed by using histopathological analysis after staining the epithelial tissue with alcian blue. Possible cytotoxicities of PGGT in vitro were assessed by examining LDH release from RTSE cells and the rate of survival and proliferation of NCI-H292 cells. In vivo liver and kidney toxicities of PGGT were evaluated by measuring serum GOT/GPT activities and serum BUN/creatinine concentrations of rats after administering PGGT orally. Results (1) PGGT did not affect in vitro mucin secretion from cultured RTSE cells. (2) PGGT significantly inhibited PMA-, EGF-, and TNF-${\alpha}$-induced MUC5AC mucin productions and the expression levels of MUC5AC mRNA from NCI-H292 cells. (3) PGGT decreased the amount of intraepithelial mucosubstances and showed the tendency of expectorating airway mucus already produced. (4) PGGT increased LDH release from RTSE cells. However, PGGT did not show in vivo liver and kidney toxicities and cytotoxicity to NCI-H292 cells. Conclusion The result from this study suggests that PGGT can regulate the production and gene expression of airway mucin observed in diverse respiratory diseases accompanied by mucus hypersecretion and do not show in vivo toxicity to liver and kidney functions after oral administration. Effect of PGGT with their components should be further studied using animal experimental models that reflect the diverse pathophysiology of respiratory diseases through future investigations.

• IMMUNE NETWORK
• /
• 제11권2호
• /
• pp.107-113
• /
• 2011

Background: Metabolic disorders, including type II diabetes and obesity, present major health risks in industrialized countries. AMP-activated protein kinase (AMPK) has become the focus of a great deal of attention as a novel therapeutic target for the treatment of metabolic syndromes. In this study, we evaluated whether dietary aloe could reduce obesity-induced inflammation and adipogenesis. Methods: Male C57BL/6 obese mice fed a high-fat diet for 54 days received a supplement of aloe formula (PAG, ALS, Aloe QDM, and Aloe QDM complex) or pioglitazone (PGZ) and were compared with unsupplemented controls (high-fat diet; HFD) or mice fed a regular diet (RD). RT-PCR and western blot analysis were used to quantify the expression of obesity-induced inflammation. Results: Aloe QDM complex downregulated fat size through suppressed expression of scavenger receptors on adipose tissue macrophages (ATMs) compared with HFD. Both white adipose tissue (WATs) and muscle exhibited increased AMPK activation through aloe supplementation, and in particular, the Aloe QDM complex. Obesity-induced inflammatory cytokines (IL-$1{\beta}$ and -6) and $HIF1{\alpha}$ mRNA and protein were decreased markedly, as was macrophage infiltration by the Aloe QDM complex. Further, the Aloe QDM complex decreased the translocation of NF-${\kappa}B$ p65 from the cytosol in the WAT. Conclusion: Dietary aloe formula reduced obesity-induced inflammatory responses by activation of AMPK in muscle and suppression of proinflammatory cytokines in the WAT. Additionally, the expression of scavenger receptors in the ATM and activation of AMPK in WAT led to reduction in the percent of body fat. Thus, we suggest that the effect of the Aloe QDM complex in the WAT and muscle are related to activation of AMPK and its use as a nutritional intervention against T2D and obesity-related inflammation.