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The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide- binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.

사람의 간에 존재하는 cytochrome P450 (CYP450)은 약물 대사에 관여하는 중요한 효소이다. 따라서 미국 FDA 와 EU EMA 가이드라인에서는 CYP450 유도능 평가 시 효소활성측정법과 더불어 유전자발현측정법을 이용할 것을 권장하고 있다. 본 연구에서는 LC-MS/MS와 qRT-PCR을 이용하여 폐동맥 고혈압 치료제인 bosentan의 CYP450 동질 효소(CYP1A2, 2B6, 3A4)에 대한 유도능을 평가하였다. Bosentan에 의해 478과 923 두 도너의 CYP3A4 mRNA 발 현량이 크게 증가하였고, 효소활성에 비해 923 도너의 유도능이 10배 이상 증가하였다. CYP2B6에서도 478 도너에 서 mRNA 발현량이 크게 증가하였다. 효소활성측정법은 복잡한 분석 방법과 LC-MS/MS 장비를 필요로 하나, 유전 자발현측정법은 실험과정이 간편하고, 민감도가 높으며 mRNA 발현량 변화를 직접적으로 확인할 수 있다는 장점을 가지고 있다. 그러므로, 유전자발현측정법은 CYP450 유도능 평가에 유용한 측정법으로 이용될 수 있을 것으로 기대 된다.

Human liver cytochrome P450 (CYP450) isozymes are principle drug-metabolizing enzymes. The US FDA and EU EMA guidelines suggest that along with enzyme activity measurements, mRNA expression levels could be used to evaluate CYP450 enzyme induction. We evaluated the potential induction ability of Bosentan, as a pulmonary arterial hypertension drug, on CYP450 isozymes (CYP1A2, 2B6 and 3A4) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantitative real-time polymerase chain reaction (qRT-PCR). Bosentan treatment increased CYP3A4 mRNA expression in HMC478, HMC923 cell, and it were at least 10 times higher than enzyme activity of that. CYP2B6 mRNA expression was induced in HMC478. CYP450 metabolism was established In vitro by determining enzymatic activity using LC-MS/MS which involves a complex method and utilizes expensive equipment. However, qRT-PCR is a simple and sensitive method for monitoring changes in mRNA expression. Therefore, we thought that mRNA expression measurement is a useful tool for detection of CYP450 isozymes induction.

This study was conducted to compare the mRNA expression levels of myogenic-adipogenic makers in the skeletal muscle and adipocytes formation, body weight, rumen weight, and papilla length on Hanwoo calves at newborn and 6 months of age. Animals used three newborn Hanwoo calves (NC) and three Hanwoo calves 6 months of age (SC). Body weight and rumen weight were significantly increased in SC compared to NC (p < 0.01), and papilla length was longer about 10-fold in SC than NC. Adipocytes was possible to visually identify more adipocytes in SC compared to NC, and were mainly formed around the blood vessels. mRNA expression of myogenin, myosin heavy chain 1 and myosin heavy chain 2A in both longissimus dorsi (LD) and semimembranosus (SM) was found to increase with calves growth (p < 0.01), and it was confirmed that have higher levels of mRNA expression in SM than LD. In LD tissues, the mRNA expression of stearoyl-CoA desaturase (SCD, p < 0.03) and peroxisome proliferator activated receptor γ (PPARγ, p < 0.04) was significantly higher in SC than NC. In SM tissues, mRNA expression levels of SCD (p < 0.02) and CCAAT/enhancer binding protein β (C/EBPβ, p < 0.01) were higher in SC than NC, and also mRNA expression levels of PPARγ increased, but there was no significant difference. Thus, the calves period suggests that it is an important step in the development of the rumen and the myogenesis and adipogenesis.

Ski protein is a nuclear transcription factor that does not bind DNA directly. Due to its unique binding properties with multiple factors, Ski could perform various roles in the regulation of both cellular proliferation and differentiation. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein isabsent in granulosa cells of preovulatory follicle, its mRNA(c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by real time-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and post ovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.

Plasminogen activators system (PAs) is one of important physiological system for reproductive events such as oocyte maturation, acrosome reaction, fertilization, and uterine remodeling. It is consist of two-types of PAs (urokinase-type, uPA; tissue-type, tPA), uPA-specific receptor (uPAR), and type-1 PA inhibitor (PAI-1). In various type of cells, activity of these system was regulated by gonadotropin, cytokines, and hormones, however, regulation of mRNA expression in porcine uterine cells was not clear. Therefore, this study aimed to investigate the effect of progesterone (P4) on mRNA expression of uPA, tPA, uPAR, and PAI-1 in epithelial cells of porcine uterine endometrium. Epithelial cells were isolated from luminal epithelium in endometrium and cultured at 80% confluence. Then, it were subsequently incubated with different concentration of P4 (0, 0.1, 1, 5, 10, and 20 ng/mL) for 5 min and 24 h at 38.5℃, 5% CO2 in air. mRNA expression was measured by reverse-transcription PCR method. In results, expression of uPA mRNA in P4-exposed epithelial cell for 5 min was increased by 0.1 and 20 ng/mL P4 treatment. However, 1, 5, and 10 ng/mL P4 reduced uPA mRNA expression compared to 0.1 ng/mL treatment (p<0.05). On the other hand, all P4 treatment groups for 24 h enhanced mRNA expression of uPA. tPA mRNA in cultrued cells with P4 for 5 min and 24 h was enhanced by 10 and 20 ng/mL treatment, whereas 5 ng/mL P4 reduced. And 20 ng/mL P4 treatment increased uPAR mRNA expression in epithelial cells that were incubated for 5 min and 24 h. Similar to result of tPA mRNA, PAI-1 expression was enhanced by 5, 10, and 20 ng/mL treatment groups. The findings in this study show that mRNA expression of PAs system components were differently regulated by P4 in porcine uterine cells and further study regarding to regulation of this system was needed.

Changes of gonadal morphology and mRNA expression patterns of vitellogenin were investigated in Siberian sturgeon Acipenser baerii (Chondrostei) during its early gonadal maturation period. Early differentiations and morphological transitions of both ovaries and testes appeared to occur actively until the age of 3 years, however from then on, the maturation patterns to full maturity were largely gender-dependent, in which males showed a faster progression of maturation than did females while females experienced a steady-state progress with a lagged interval before entering the final maturation. Expression of vitellogenin mRNAs are closely correlated with transitional patterns of gonadal appearances. In both females and males, hepatic mRNA levels of vitellogenin exponentially increased in the earliest interval (up to 1-year-old). However, in subsequent periods, vitellogenin expression in females continued to increase with age, whereas in males, the expression stabilized at a younger age. Nevertheless, at the age older than or equal to 7-year-old, fully matured individuals showed a quite low level of vitellogenin expression in both females and males. Collectively, results from this study could be useful as a fundamental guideline to address the gonad maturation of this sturgeon species, which is helpful for making practical decisions about farming practices and management for caviar production on local sturgeon farms.

As an one of multifunctional cytokine, transforming growth factor-β (TGF-β) induces various signaling pathway in cells via bind to their receptor. This action of TGF-β plays a crucial role in regulation of uterine environment for successful pregnancy in mammalians. Plasminogen activators system (PAs) is one of important physiological system for reproductive events such as oocyte maturation, acrosome reaction, fertilization, and uterine remodeling. It is consist of two-types of PAs (urokinase-type, uPA; tissue-type, tPA), uPA-specific receptor (uPAR), and type-1 PA inhibitor (PAI-1). In various type of cells, activity of these system was regulated by gonadotropin, cytokines, and hormones. Although both of TGF-β and PAs system play important role in uterine environment, relationship between TGF-β and PAs system was not fully understood. Therefore, this study was conducted to investigate regulation of PAs activity and mRNA expression by TGF-β in porcine uterine epithelial cells (pUECs). pUECs were isolated from uterine horn and cultured until 50% confluence. Then, cells were starved in serum-free medium for 24 hours and subsequently medium supplemented with TGF-β supplemented medium (0, 0.1, 10, 50, and 100 ng/mL) for 24 hours. The expression level of mRNA was analyzed using real-time PCR and PA activity assay was used for measurement of PA activity in supernatant. In results, PA activity was significantly decreased in TGF- β 10, 50, and 100 ng/mL treared group compared with control group (p<0.05). On the other hand, TGF-β did not affect to expression levels of uPA, tPA, uPAR and PAI-1 mRNA. In present study, although expression of uPA, tPA, uPAR, and PAI-1 mRNA were not influenced by TGF-β treatment, relative PA activity was reduced according to TGF-β concentration increased. Therefore, these results suggested that TGF-β regulate translation process for control the PA activity in porcine uetrine cells.

목적: 본 연구에서는 비타민 C 유효성분을 포함하여 독자적인 기술로 제조한 리포좀 형태인 '네오젠 브이 바이옴 리포좀 TM(NEOGEN V.BIOME LIPOSOMETM)'을 인간진피섬유아세포(normal human dermal fibroblasts, NHDF)에 적용하여 콜라겐 발 현 유전자 COL1A1 및 히알루론산 합성효소 유전자 HAS 2에 대한 세포효능을 평가하여 기능성화장품으로 활용가능성을 확인하 고자 한다. 방법: 인간진피섬유아세포에서 '네오젠 브이 바이옴 리포좀™(NEOGEN V.BIOME LIPOSOMETM)' 처리에 의한 세포생 존율과, 정량적 qRT-PCR분석을 통해 콜라겐 발현 유전자 COL1A1 및 히알루론산 합성효소 유전자 HAS2 발현량을 분석하였다. 결과: 세포독성평가 결과, '네오젠 브이 바이옴 리포좀TM (NEOGEN V.BIOME LIPOSOME™)'의 인간진피섬유아세포에 대한 세포 독성은 0.25% 이하에서 관찰되지 않았다. 시험 물질을 0.25% 이하 농도(0.05, 0.1, 0.25%)로 처리 시 COL1A1 mRNA 및 HAS2 발현이 음성대조군 대비 농도의존적으로 증가되는 것을 확인하였다. 결론: '네오젠 브이 바이옴 리포좀TM (NEOGEN V.BIOME LIPOSOMETM)'은 인간진피섬유아세포 내 COL1A1 mRNA 및 HAS2 mRNA 발현 촉진으로 콜라겐 생성 및 히알루론산 합성효소 생 성 증가에 도움을 주는 것으로 주름개선 기능성 화장품 소재로 적용 가능하다는 것을 시사한다.

Purpose: This study investigated functional cosmetic agent for anti-wrinkle effects of 'NEOGEN V.BIOME LIPOSOME™', which is a liposome in the form of a proprietary technology including a vitamin C active ingredient, on human dermal fibroblasts (HDFs). Methods: The cytotoxicity of 'NEOGEN V.BIOME LIPOSOME™' was measured by performing a water-soluble tetrazolium salt (WST-1) assay. The relative mRNA expression of collagen (COL1A1 ) and hyaluronan synthase (HAS2 ) was measured by quantitative real-time PCR (qRT-PCR) assay. Results: HDF cell viability of 'NEOGEN V.BIOME LIPOSOME™' was no cytotoxic effect at concentrations below 0.25%. The expression of COL1A1 and HAS2 mRNA was increased in a concentration-dependent manner compared with the negative control group. Conclusion: “NEOGEN V.BIOME LIPOSOME™“ helps to increase collagen and hyaluronan synthase production by promoting COL1A1 and HAS2 mRNA expression in HDFs; therefore, it is expected to function as an anti-wrinkle cosmetic material.

目的: 本研究调查了功能性美容剂“NEOGEN V.BIOME LIPOSOME™”对人真皮成纤维细胞 (HDFs)的抗皱效果。 这是一种采用专利技术形式 含有维生素 C 活性成分的脂质体。方法: “NEOGEN V.BIOME LIPOSOME™”的细胞 毒性通过水溶性四唑盐(WST-1)测定法进行测定。通过定量实时PCR(qRT-PCR) 测定法测量胶原蛋白 (COL1A1 ) 和乙酰透明质酸合酶 (HAS2 ) 的mRNA 表达。结果: “NEOGEN V.BIOME LIPOSOME™”对HDFs细胞生存率测定 结果，在浓度低于0.25%时没有细胞毒性。与阴性对照组相比，COL1A1 和HAS2 mRNA的表达呈浓度依赖性增 加。结论: “NEOGEN V.BIOME LIPOSOME™”通过促进 HDFs细胞中 COL1A1 和HAS2 mRNA的表达，有助于增 加胶原蛋白和透明质酸合酶的产生；因此，有望作为抗皱化妆品原料发挥作用。

CCAAT/enhancer binding protein(C/EBP)는 지방전구세포의 분화과정에서 발현하는 전사인자군에 속한다. 이러한 C/EBP 중에서 C/EBPα는 지방세포의 분화와 지방침착에 있어 중요한 역할을 수행하는 것으로 알려져 있다. 본 연구에서는 한우 C/EBPα 유전자를 분리 동정하고 그 발현을 조사하였다. 한우 C/EBPα 유전자는 1059bp의 open reading frame으로 구성되어 있으며 353개의 아미노산을 암호화하고 있었다. 그리고 한우 C/EBPα 아미노산을 다른 종과 비교하였을 때 매우 높은 상동성을 나타내었다. Northern blotting 분석에 의하여 12개월 한우에 있어서 C/EBPα mRNA의 분석을 실시한 결과 지방조직에서 가장 높은 발현을 나타내었으며 대장과 폐에서 매우 낮은 발현을 확인할 수 있었다. 또한 12개월, 26개월, 30개월 한우의 등심과 지방에 있어서 C/EBPα 유전자의 발현을 real-time RT-PCR로 분석한 결과 등심에서는 26개월에서 가장 높은 발현을 보였으며 지방에서는 12개월과 26개월에서 높은 발현을 나타내었다.

CCAAT/enhancer binding proteins(C/EBP) are a group of transcription factors expressed during preadipocyte differentiation. In the C/EBPs, C/EBPα plays an important role in lipid deposition and adipocyte differentiation. In this studies, we report the identification, characterization, and expression of a Hanwoo C/EBPα. The Hanwoo C/EBPα DNA includes a 1059 bp open reading frame encoding a protein of 353 amino acids. The C/EBPα amino acid sequences of the Hanwoo show strong conservation with the corresponding sequences reported in other species. The distribution of C/EBPα mRNA in various tissues of Hanwoo aged 12 months were investigated using Northern blotting analysis. The highest expression was detected in adipose tissue and more lower expression was detected in colon and lung. We also identified expression of C/EBPα mRNA in Hanwoo sirloin and adipose tissue aged 12, 26, and 30 months by real-time RT-PCR. The higest expression were detected at 26 months in the sirloin and at 12 and 26 months in the adipose tissue.

지방세포분화 과정에서 중추적인 역할을 한다고 보고되어지고 있는 PPARγ 유전자를 cloning 하기 위하여 한우 12개월 지방조직을 이용하여 Total RNA를 추출하고 RT-PCR을 수행하여 1515bp, 505 아미노산 서열을 가진 PPARγ 유전자를 cloning하였다. bovine을 제외한 다른 4종의 다른 동물과는 아미노산 서열을 비교한 결과 모두 90% 이상의 상동성을 나타내었다. 특히 NCBI에 보고된 Norwegian cattle PPARγ (NCBI Accession No. O18971)의 아미노산 서열과 비교한 결과 99.2%의 상동성을 나타내었으며 아미노산 서열중 81번째 이소루신, 140번째 아르기닌, 157번째 글루탐산, 486번째 리신이 각각 트레오닌, 트립토판, 글리신, 이소루신으로 치환된 상태의 아미노산 서열을 나타내었다. 특히 nuclear hormone receptor의 DNA binding domain 영역에 포함되는 140번째, 157번째 아미노산의 치환은 앞으로 한우 PPARγ 유전자가 조절하는 다른 유전자와의 관계를 밝히는데 매우 중요한 점이 될 것으로 사료되어진다. 한우 PPARγ의 발현은 지방조직에서 매우 높은 발현을 보였으며, 한우 12개월령과 30개월령 지방조직에서의 PPARγ 발현을 비교분석하였을 때 12개월령에서보다 30개월령 지방조직에서 PPARγ 발현이 약 6배정도 높았다. 이와 같은 결과는 한우 PPARγ 유전자도 사람과 생쥐에서와 같이 지방분화에 관여하고 있다는 보고와 일치하는 결과라고 사료된다.

The peroxisome proliferator-activated receptor γ(PPARγ), a member of the steroid/thyroid nuclear hormone receptor suferfamily of ligand-activated transcription factor, is an important regulator of adipocyte gene expression and differentiation. In this studies, we report the identification, characterization, and expression of a Hanwoo PPARγ gene. The PPARγ cDNA sequence of the Hanwoo show strong conservation with the corresponding sequences reported in other species except of three amino acid sequences. The distribution of PPARγ mRNA in various tissues of Korean cattle aged 12 months were investigated using Northern Blot analysis. The highest expression was detected in adipose tissue, more lower expression was detected in colon, small intestine, kidney, lung, while expression was not detected in brain, heart. PPARγ expression was higher in adipose tissue of Korean cattle when aged 30 months than aged 12 months. These results indicated PPARγ, regulator adipocyte gene expression and differentiation, related on adipose differentiation in Korean native cattle(HANWOO).