This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (～35 kDa), dimer (～70 kDa), trimer (～105 kDa) and hexamer (～210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (～300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher’s study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (β- MeOH), anionic detergent (SDS) and heat (95℃) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

Linuron is a pesticide with a weak anti-androgenic property, which impacts male reproductive organs. In this study, to clarify whether linuron affects the cellular antioxidant system of ventral prostate, gene expression patterns of the representative antioxidant enzymes such as glutathione peroxidase (GPx), selenoprotein P (SePP), and superoxide dismutase (SOD) were investigated in the rat ventral prostates exposed to linuron using real-time RT-PCR analyses. Sprague-Dawley rats castrated at 6 weeks old were treated with linuron (25, 50, or 100 mg/kg per oral) daily for 10 days after testosterone propionate administration (0.4 mg/kg) subcutaneously. As compared to normal control animals, mRNA levels of phospholipid hydroperoxide GPx (PHGPx), SePP, and Mn SOD significantly increased in the prostates exposed to linuron (25, 50, and 100 mg/kg). However, cytosolic GPx (100 mg/kg) and Cu/Zn SOD (25, 50, and 100 mg/kg) mRNA levels significantly decreased in the ventral prostates. These results indicate that linuron upregulates the expressions of PHGPx, SePP, and Mn SOD mRNAs, but down-regulates the expressions of cytosolic GPx and Cu/Zn SOD in rat prostates, suggesting that linuron may have dual effects in the cellular antioxidant system of prostate.

Odontogenic cysts are classified into inflammatory and developmental origins. The most common representative inflammatory cyst is periapical cyst and the most common representative developmental cyst is dentigerous cyst and cyst which show character of tumor is odontogenic keratocyst and cyst of which cystic epithleial lining cells transform to ameloblastoma is unicystic ameloblastoma. About ten years ago p63 protein that are closely related to p53 protein was found. Authors studied about comparative pattern of expression of p63 protein in periapical cyst, dentigerous cysts, odontogenic kertocysts and unicystic ameloblastomas. Authors selected 10 cases for every four types of cyst and performed immunohistochemical staining by using monoclonal antibody about p63 protein, LSAB(labelled streptoavidin biotin) reactant and HRP(horse raish peroxidase) system. Positive cells about p63 protein were expressed at basal layer of cystic lining epithelium in periapical cysts, odontogenic keratocysts and unicytic ameloblastomas. On the contrary, in dentigerous cysts positive cells were expressed at surfce layer. Perapical cysts and odontogenic keratocysts showed significantly high values of labelling indices.(periapical cyst:72.49%, odontogenic keratocyst:64.72%, dentigerous cyst:8.94%, unicystic ameloblastoma: 5.25%) Odontogenic keratocyst showed the most strong staining intensity and the second was periapical cyst, the third was dentigerous cyst, and lastly unicystic ameloblastoma. Conclusively cause that the positive cells appeared at surface layer in dentigerous cyst reflected the position of epithelium to the enamel, and labelling indices of p63 protein were closely related to proliferative capacity and intensity of expression closely related to the labelling index and thus labelling index was also closely related to proliferative capacity of cystic lining epithelium.

Palatal development is one of the crucial events in craniofacial morphogenesis, according to the significant signaling pathway including the out growth, elevation, and fusion of palatal shelves. In the fusion of palatal shelves, epithelial to mesenchymal transition (EMT) is a fundamental process to achieve the proper morphogenesis of palate. Mechanisms of EMT have been reported as the processes of migration, apoptosis or general EMT through the modulations through various signalling molecules. Rgs19, known as a regulator of G protein signaling (RGS) family through GTPase activity, showed the interesting epithelial expression patterns in various organogeneses including the significant expression patterns of Rgs19 in palatal development. To evaluate the precise function of Rgs19 in palatogenesis, we employed the gain and loss of function studies using ASODN treatments and gene electroporations while in vitro palate organ cultivations. Knockdown of Rgs19 using treatments of AS-ODN showed the retarded palatal fusion with the decreased patterns of apoptosis in mesial epithelium edge (MEE). In addition, alteration patterns of related genes were examined with the qRT-PCR. And epithelial mesenchyme transition (EMT) process was delayed in medial edge epithelium (MEE) throught immunohistochemistry of pancytokeratin, which known as epithelial cell marker. Morphological changes were observed with the three dimensional reconstruction method. These results show that expression of Rgs19 in MEE has crucial role of EMT, also Rgs19 affects to palatal fusion by regulation of apoptosis through the signalling modulations.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

An efficient transient expression system has been developed and characterized for the production of foreign genes in seedlings. The seedlings can be easily produced from commercial seeds used for vegetable sprouts. In principal, a chemical abrasive was employed to generate wounds in seedlings prior to vacuum-infiltration with Agrobacterium tumefaciens bearing the target gene. This optimized chemical wounding-assisted agro-infiltration process resulted in up to 15-fold increase in β -glucuronidase (GUS) enzyme activity. This procedure has been used efficiently to express hepatitis B surface antigen (HBsAg) protein in a transient mode. Therefore, seedlings with proper wounds can be suggested as a convenient tool for the production of useful recombinant proteins.

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

Glutathione (GSH)은 직접적으로 활성산소종을 제거하거나 GSH peroxidase와 같은 활성산소종 제거 효소의 조효소로써, 산화적 스트레스로부터 세포를 방어하는 데 중요한 역할을 한다. GSH peroxidase는 두 분자의 GSH을 이용해 세포 내 과산화수소를 물로 전환한다. N-acetyl-L cysteine (NAC)는 항산화제 중 하나로 세포 내 GSH의 전구물질로 이용된다. 본 연구는, 0mM에서 20mM의 NAC 단독 처리

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH–quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results shown that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, I will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

The study investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and gene expression. Three experiments were conducted. Firstly the trans-ε-viniferin was purified from the leaves and stems of the Vitis amurensis , a common wild grape found in Korea, Japan, and China. In the first experiment, a total of 594 cumulus oocytes complexes (COCs) were used for the evaluation of the nuclear maturation. COCs were matured with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM). After IVM 42 44 h, the nuclear maturation was evaluated. In the second experiment, a total of 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular GSH and ROS levels. In the third experiment, the gene expression of oocytes matured with trans-ε-viniferin (0.5 μM) and the untreated group were evaluated after IVM. As results, we observed that trans-ε- viniferin treatment during IVM did not improve the nuclear maturation. But significantly increased (p<0.05) intracellular GSH levels in 0.5 μM group (0 μM vs. 0.5 μM; 14.6 vs. 16.8 pmol/oocyte) and reduced ROS levels (0 μM vs. 0.5 μM and 50 μM; 174.6 vs. 25.7 and 23.8 pixel/oocyte). The trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (p<0.05) expression of Dnmt1 mRNA in 0.5 μM treatment group than in the control group. But, the other gene expressions (PCNA, OCT4, caspase3, BAK, BAX and sit1) did not significantly differ from the control. In conclusion, these results indicated that the trans-ε-viniferin treatment during porcine IVM increased the cytoplasmic maturation through increasing the intracellular GSH synthesis, reducing ROS levels and increasing the Dnmt1 gene expression.

Macrophages can recognize antigens and microorganisms, and then initiate an appropriate defense. However, there has been a lack of comprehensive information regarding the genes that are modulated by commensal yeasts, including Saccharomyces cerevisiae or Saccharomyces exiguus. In addition, it is not clear to what extent the beneficial yeasts modulate the immune response against microbes and/or microbial toxins. Using DNA microarray, which contains approximately 25,000 genes, we studied interactions between host cells and yeast/bacterial toxin (LPS) by analyzing the transcriptional response of macrophages stimulated by Saccharomyces exiguus and/or Lipopolysaccharides. Thirty three genes were identified to be modulated by more than two folds between groups of macrophage cells. Pathway analysis provided insight into the mutual interactions. Of particular interest was the responses elicited by fungus in murine macrophage cells, including modulation of immunity/defense, cellular signal transduction, cell proliferation/differentiation, and transport. This finding indicates that the yeast induces immune response pathways as well as those associated with cell proliferation and transport. Among the 33 genes identified from the DNA microarray screening, eight genes were further checked by RT-PCR analysis using gene specific primers. Compared to those of negative control, sequential treatment with the yeast strain followed by LPS apparently induced expression of Tnfaip3, IL7R, and CD86, while it inhibited expression of Cxcl10 and CD83. In conclusion, this study identified the genes that are up-regulated by Saccharomyces exiguus. A further study is needed in order to determine whether these genes are modulated at the protein level, and also for their roles in control of immune responses.

Human growth hormone (hGH), one of the most important hormones in medicine, is secreted from anterior pituitary gland. Its broad physiological function includes body growth, cell regeneration, increasement of muscle volume, bone density, body fat reduction, and so on. Due to the wide range of therapeutic effects, the hGH produced from E. coli has been commercialized already. In this study, we asked whether it is possible to produce recombinant hGH efficiently from various cultured mammalian cells. To meet this purpose, we chose a retrovirus vector system for transfer and expression of the hGH gene in various mammalian cells. Analyses of RT-PCR, ELISA, and Western blot to determine expression of the hGH gene showed the highest production of the hGH was determined from chicken embronic fibroblast (CEF) cells with the concentration of 8.58 μg/ml. The biological activity of the hGH was similar to the commercially available counterpart. These results suggest that mass production of hGH is possible not only in the E. coli but also in the various mammalian cells.

The in vitro maturation rate of vitrified-thawed canine oocytes was 30.8±3.4%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (52.0±2.5%, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were 17.5±2.5% and 8.8±3.4%, respectively. This results were lower than the control group (43.6±3.2% vs 20.0±3.0%). SOD1 gene expression of 1～2 mm of follilce size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1～2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1～2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3’- and 5’-RACE PCR and RT- PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.