• Tuberculosis and Respiratory Diseases
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• v.64 no.4
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• pp.285-292
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• 2008

Background: A diagnosis of malignant pleural effusion is clinically important, as the prognosis of lung cancer patients with malignant pleural effusion is poor. The diagnosis will be difficult if a cytological test is negative. This study was performed to investigate whether the detection of hypermethylation of the p16 (CDKN2A) and retinoic acid receptor b2 (RARB2) genes in pleural fluid is useful for a diagnosis of malignant pleural effusion. Methods: Pleural effusion was collected from 43 patients and was investigated for the aberrant promoter methylation of the RARB2 and CDKN2A genes by use of methylation-specific PCR. Results were compared with findings from a pleural biopsy and from pleural fluid cytology. Results: Of 43 cases, 17 cases of pleural effusion were due to benign diseases, and 26 cases were from lung cancer patients with malignant pleural effusion. Hypermethylation of the RARB2 and CDKN2A genes was not detected in the case of benign diseases, independent of whether or not the patients had ever smoked. In 26 cases of malignant pleural effusion, hypermethylation of RARB2, CDKN2A or either of these genes was detected in 14, 5 and 15 cases, respectively. The sensitivities of a pleural biopsy, pleural fluid cytology, hypermethylation of RARB2, hypermethylation of CDKN2A, or hypermethylation of either of the genes were 73.1%, 53.8%, 53.8%, 19.2%, and 57.7%, respectively; negative predictive values were 70.8%, 58.6%, 58.6%, 44.7%, and 60.7%, respectively. If both genes are considered together, the sensitivity and negative predictive value was lower than that for a pleural biopsy, but higher than that for pleural fluid cytology. The sensitivity of hypermethylation of the RARB2 gene for malignant pleural effusion was lower in small cell lung cancers than in non-small cell lung cancers. Conclusion: These results demonstrate that detection of hypermethylation of the RARB2 and CDKN2A genes showed a high specificity, and sensitivity was higher than for pleural fluid cytology. With a better understanding of the pathogenesis of lung cancer according to histological types at the molecular level, and if appropriate genes are selected for hypermethylation testing, more precise results may be obtained.

• Journal of Korean Society of Forest Science
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• v.87 no.2
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• pp.164-172
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• 1998

This study was conducted to find the optimum transformation condition using Agrobacterium harboring promoterless GUS gene. The optimal medium for shoot induction from leaves of Populus nigra${\times}$P. maximowiczii was MS medium supplemented with $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP(94% regeneration frequency and 11.5 average number of shoot) According to the test using pBI121, the concentration of antibiotics for selection marker gene was $100mg/{\ell}$ kanamycin or $60mg/{\ell}$ geneticin in the SIM(shoot inducing medium) 3. Two weeks later, callus was induced in the SIM 3 and this callus grew up to 0.5-1cm shoots after 6 weeks in the new SIM 3. And the treatment with methylation inhibitor(5-azacytidine) led to a dramatic increase in foreign gene expression rate from 5.7% to 26.7%. The vector systems showed. different transformation efficiencies based on the fluorometric and histochemical GUS assay. In this study the vector systems used for transformation seemed to affect transformation frequency, in which pEHA101 yielded more transformants(35.9%) than LBA4404/pBI121 did(5.7%). This result indicated that pEHA101 was effective to insert the promoterless foreign gene into a poplar genome.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.1-35
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• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5941-5948
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• 2013

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.12
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• pp.1695-1704
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• 2014

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1837-1847
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• 2020

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.

• Journal of Life Science
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• v.29 no.9
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• pp.1047-1054
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• 2019

Transposable elements (TEs) occupy approximately 45% of the human genome and can enter functional genes randomly. During evolutionary radiation, multiple copies of TEs are produced by duplication events. Those elements contribute to biodiversity and phylogenomics. Most of them are controlled by epigenetic regulation, such as methylation or acetylation. Every species contains their own specific mobile elements, and they are divided into DNA transposons and retrotransposons. Retrotransposons can be divided by the presence of a long terminal repeat (LTR). They show various biological functions, such as promoter, enhancer, exonization, rearrangement, and alternative splicing. Also, they are strongly implicated to genomic instability, causing various diseases. Therefore, they could be used as biomarkers for the diagnosis and prognosis of diseases such as cancers. Recently, it was found that TEs could produce miRNAs, which play roles in gene inhibition through mRNA cleavage or translational repression, binding seed regions of target genes. Studies of TE-derived miRNAs offer a potential for the expression of functional genes. Comparative analyses of different types of miRNAs in various species and tissues could be of interest in the fields of evolution and phylogeny. Those events allow us to understand the importance of TEs in relation to biological roles and various diseases.

• Journal of Life Science
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• v.30 no.12
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• pp.1092-1100
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• 2020

The six-transmembrane epithelial antigen of prostate 4 (Steap4) is a metalloreductase that plays a role in intracellular iron and cupper homeostasis, inflammatory response, and glucose and lipid metabolism. Previously, Steap4 has been reported to stimulate adipocyte differentiation; however, the underlying mechanisms of this action remain unexplored. In the present study, we investigated the molecular mechanisms involved in Steap4-induced adipocyte differentiation using 3T3-L1 cells, immortalized brown adipocyte (iBA) cells, and mouse embryonic fibroblast C3H10T1/2 cells. The knockdown of Steap4 using adenovirus-containing shRNA attenuated mitotic clonal expansion (MCE), as evidenced by the impaired proliferation of 3T3-L1 cells, iBA cells, and C3H10T1/2 cells within 48 hr after adding the differentiation medium. Steap4 knockdown downregulated G1/S phase transition-related cell cycle regulators (including cyclin A and cyclin D) and upregulated cell cycle inhibitors (including p21 and p27). Furthermore, Steap4 knockdown inhibited the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and Akt. Moreover, Steap4 knockdown repressed the expression of early adipogenic activators, such as CCAAT-enhancer-binding protein β (C/EBPβ) and Kruppel-like factor family factor 4 (KLF4). On the other hand, Steap4 knockdown stimulated the expression of adipogenic inhibitors, including KLF2, KLF3, and GATA2. The overexpression of Steap4 using an adenovirus removed the repressive histone marks H3K9me2 and H3K9me3 on the promoter of C/EBPβ. These results indicate that Stepa4 stimulates adipocyte differentiation through the induction of MCE and the modulation of early adipogenic transcription factors, including C/EBPβ, during the early phase of adipocyte differentiation.

• Journal of Chest Surgery
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• v.37 no.12
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• pp.967-977
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• 2004

Background: DNA methylation is one of the important gene expression mechanisms of the cell. When cytosine of CpG dinucleotide in promotor is hypomethylated, expression of some genes that is controlled by this promoter is altered. In this study, the author investigated the effect of DNA demethylating agent, 5-aza-2'-deoxycytidine (ADC), on the expressions of cancer antigen genes, MHC and B7 in 4 lung cancer cell lines, NCIH1703, NCIH522, MRC-5, and A549. Material and Method: After treatment of cell lines, NCIH1703, NCIH522, MRC-5 and A549 with ADC (1 uM) for 48 hours, RT-PCR was performed by using the primers of MAGE, GAGE, NY-ESO-1, PSMA, CEA, and SCC antigen gene. In order to find the optimal ADC treatment condition for induction of cancer antigen, we studied the effect of ADC treatment time and dose on the cancer antigen gene expression. To know the effect of ADC on the expression of MHC or B7 and cell growth, cells were treated with 1 uM of ADC for 72 hours for FACS analysis or cells were treated with 0.2, 1 or 5 uM of ADC for 96 hours for cell counting. Result: After treatment of ADC (1 uM) for 48 hours, the expressions of MAGE, GAGE, NY-ESO-1, and PSMA genes increased in some cell lines. Among 6 MAGE isotypes tested, and gene expression of MAGE-1, -2, -3, -4 and -6 could be induced by ADC treatment. However, CEA gene expression did not change and SCC gene expression was decreased by ADC treatment. Gene expression was generally induced 24 - 28 hours after ADC treatment and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC ADC teatment, and expression of MAGE, GAGE, and NY-ESO-1 was maintained at least 14 days after ADC teatment in ADC-Free medium. Most gene expression could be induced at 0.2 uM of ADC, but gene expression increased dependently on ADC treatment dose. The expression of MHC and B7 was not increased by ADC treatment in all four cell lines, and the growth rate of 4 cell lines decreased significantly with the increase of ADC concentrations. Conclusion: Treatment of lung cancer cell lines with ADC increases the gene expression MAGE, GAGE and NY-ESO-1 that are capable of induction of cytotoxic T lymphocyte response. We suggest that treatment with 1 uM of ADC for 48 hours and then culturing in ADC-free medium is optimal condition for induction of cancer antigen. However, ADC has no effect on MHC and B7 induction, additional modification for increase of expression of MHC, B7 and cytokine will be needed for production of efficient cancer cell vaccine.