• Archives of Pharmacal Research
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• v.11 no.1
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• pp.7-13
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• 1988

The yeast cytochrome c gene has been recloned, and the resulting cytochrome c mRNA has been translated in rabbit reticulocyte lysate translation system. The newly synthesized apocytochrome c could be methylated by exogenously added cytochrome c-lysine N-methyltransferase. Enzymatic methylation of in vitro synthesized apocytochrome c was found to facilitate specifically its import into mitochondria of yeast, but not of rat liver.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.11
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• pp.1532-1539
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• 2014

Although growth rate is one of the main economic traits of concern in pig production, there is limited knowledge on its epigenetic regulation, such as DNA methylation. In this study, we conducted methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) to compare genome-wide DNA methylation profile of small intestine and liver tissue between fast- and slow-growing weaning piglets. The genome-wide methylation pattern between the two different growing groups showed similar proportion of CpG (regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence) coverage, genomic regions, and gene regions. Differentially methylated regions and genes were also identified for downstream analysis. In canonical pathway analysis using differentially methylated genes, pathways (triacylglycerol pathway, some cell cycle related pathways, and insulin receptor signaling pathway) expected to be related to growth rate were enriched in the two organ tissues. Differentially methylated genes were also organized in gene networks related to the cellular development, growth, and carbohydrate metabolism. Even though further study is required, the result of this study may contribute to the understanding of epigenetic regulation in pig growth.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.1833-1836
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• 2012

Objective: Promoter methylation, which can be regulated by MTHFR activity, is associated with silencing of genes. In this study we evaluated the methylation status (type) of the BRCA2 promoter in ovarian cancer patients carrying different genotypes of the MTHFR gene (A or C polymorphisms at position 1298). Methods: The methylation type of the BRCA2 promoter was evaluated using bisulfate-modified DNA in methylation-specific PCR and the MTHFRa1278c polymorphism was assessed by PCR-RFLP. Results: Analysis of the BRCA2 promoter methylation type of cases showed that 7 out of 60 cases (11.7%) were methylated while the remaining 53 (88.3%) were unmethylated. In methylated cases, one out of the 7 cases had a CC genotype and the remaining 6 methylated cases had an AC genotype. The AA genotype was absent. In unmethylated cases, 34, 18, and one out of these had AC, AA and CC genotype, respectively. Conclusion: There was no significant relationship between the methylation types of the BRCA2 promoter in different genotypes of MTHFRa1298c polymorphism in ovarian cancer; p=0.255. There was no significant relation between the methylation types of the BRCA2 promoter in different genotypes of the MTHFRa1298c polymorphism in ovarian cancer.

• The Korean Journal of Nuclear Medicine Technology
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• v.22 no.2
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• pp.67-73
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• 2018

$[^{11}C]PIB$ synthesis has been performed by a loop-methylation and HPLC purification in our lab. However, this method is time-consuming and requires complicated systems. Thus, we developed an on-cartridge method which simplified the synthetic procedure and reduced time greatly by removing HPLC purification step. We compared 6 different cartridges and evaluated the $[^{11}C]PIB$ production yields and specific activities. $[^{11}C]MeOTf$ was synthesized by using TRACERlab FXC Pro and was transferred into the cartridge by blowing with helium gas for 3 min. To remove byproducts and impurities, cartridges were washed out by 20 mL of 30% EtOH in 0.5 M $NaH_2PO_4$ solution (pH 5.1) and 10 mL of distilled water. And then, $[^{11}C]PIB$ was eluted by 5 mL of 30% EtOH in 0.5 M $NaH_2PO_4$ into the collecting vial containing 10 mL saline. Among the 6 cartridges, only tC18 environmental cartridge could remove impurities and byproducts from $[^{11}C]PIB$ completely and showed higher specific activity than traditional HPLC purification method. This method took only 8 ~ 9 min from methylation to formulation. For the tC18 environmental cartridge and conventional HPLC loop methods, the radiochemical yields were $12.3{\pm}2.2%$ and $13.9{\pm}4.4%$, respectively, and the molar activities were $420.6{\pm}20.4GBq/{\mu}mol$ (n=3) and $78.7{\pm}39.7GBq/{\mu}mol$ (n=41), respectively. We successfully developed a facile on-cartridge methylation method for $[^{11}C]PIB$ synthesis which enabled the procedure more simple and rapid, and showed higher molar radio-activity than HPLC purification method.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1572-1575
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• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• BMB Reports
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• v.47 no.11
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• pp.609-618
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• 2014

DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development.

• Nutrition Research and Practice
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• v.11 no.2
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• pp.105-113
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• 2017

BACKGROUND/OBJECTIVES: A high-fat diet (HFD) induces obesity, which is a major risk factor for cardiovascular disease and cancer, while a calorie-restricted diet can extend life span by reducing the risk of these diseases. It is known that health effects of diet are partially conveyed through epigenetic mechanism including DNA methylation. In this study, we investigated the genome-wide hepatic DNA methylation to identify the epigenetic effects of HFD-induced obesity. MATERIALS AND METHODS: Seven-week-old male C57BL/6 mice were fed control diet (CD), calorie-restricted control diet (CRCD), or HFD for 16 weeks (after one week of acclimation to the control diet). Food intake, body weight, and liver weight were measured. Hepatic triacylglycerol and cholesterol levels were determined using enzymatic colorimetric methods. Changes in genome-wide DNA methylation were determined by a DNA methylation microarray method combined with methylated DNA immunoprecipitation. The level of transcription of individual genes was measured by real-time PCR. RESULTS: The DNA methylation statuses of genes in biological networks related to lipid metabolism and hepatic steatosis were influenced by HFD-induced obesity. In HFD group, a proinflammatory Casp1 (Caspase 1) gene had hypomethylated CpG sites at the 1.5-kb upstream region of its transcription start site (TSS), and its mRNA level was higher compared with that in CD group. Additionally, an energy metabolism-associated gene Ndufb9 (NADH dehydrogenase 1 beta subcomplex 9) in HFD group had hypermethylated CpG sites at the 2.6-kb downstream region of its TSS, and its mRNA level was lower compared with that in CRCD group. CONCLUSIONS: HFD alters DNA methylation profiles in genes associated with liver lipid metabolism and hepatic steatosis. The methylation statuses of Casp1 and Ndufb9 were particularly influenced by the HFD. The expression of these genes in HFD differed significantly compared with CD and CRCD, respectively, suggesting that the expressions of Casp1 and Ndufb9 in liver were regulated by their methylation statuses.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.2 no.2
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• pp.123-131
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• 2016

Increasing clinical demand for carbon-11 labeled radiopharmaceuticals has triggered technological advances in fields of radiochemistry and automated modules. Even though carbon-11 has a short half-life ($t_{1/2}=20.4min$), the consecutive second production of carbon-11 labeled radiopharmaceutical in one $^{11}C$-synthetic module should be delayed at least over 4 h to avoid the high radiation exposure. We herein aimed to produce two different carbon-11 labeled radiopharmaceuticals ([$^{11}C$]PIB and [$^{11}C$]methionine) by sharing of [$^{11}C$]methylation source in one $^{11}C$-synthetic module. The synthesis of $^{11}C$-labeling reagents ($[^{11}C]CH_3I$ or $[^{11}C]CH_3OTf$) is fully automated using the commercial TRACERlab $FX_{C-pro}$ module and is readily adaptable to $^{11}C$-labeling reactor for [$^{11}C$]PIB as well as another $^{11}C$-labeling apparatus for [$^{11}C$]methionine via the three-way valve. After completing the [$^{11}C$]PIB production, the re-synthesized $[^{11}C]CH_3I$ was passed through the three-way valve connected the polyetheretherketone (PEEK) line and loaded into the C18 Sep-Pak cartridge including the methionine precursor. The labeled product [^${11}C$]methionine was purified by a simple cartridge separation and reformulated into saline. The radiochemical yield of [$^{11}C$]PIB and [$^{11}C$]methionine were $5.3{\pm}0.6%$ and $18.7{\pm}0.8%$ (n.d.c.), respectively, with over 97% of radiochemical purity. The specific activity of [$^{11}C$]PIB was over $110GBq/{\mu}mol$. Total production time of two radiopharmaceuticals needs about 2 h from $1^{st}$ beam irradiation including quality control tests. Final [$^{11}C$]PIB and [$^{11}C$]methionine were satisfied all quality control test standards.

• Molecules and Cells
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• v.25 no.3
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• pp.358-367
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• 2008

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.

• Molecules and Cells
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• v.40 no.9
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• pp.667-676
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• 2017

Abnormal differentiation of muscle is closely associated with aging (sarcopenia) and diseases such as cancer and type II diabetes. Thus, understanding the mechanisms that regulate muscle differentiation will be useful in the treatment and prevention of these conditions. Protein lysine acetylation and methylation are major post-translational modification mechanisms that regulate key cellular processes. In this study, to elucidate the relationship between myogenic differentiation and protein lysine acetylation/methylation, we performed a PCR array of enzymes related to protein lysine acetylation/methylation during C2C12 myoblast differentiation. Our results indicated that the expression pattern of HDAC11 was substantially increased during myoblast differentiation. Furthermore, ectopic expression of HDAC11 completely inhibited myoblast differentiation, concomitant with reduced expression of key myogenic transcription factors. However, the catalytically inactive mutant of HDAC11 (H142/143A) did not impede myoblast differentiation. In addition, wild-type HDAC11, but not the inactive HDAC11 mutant, suppressed MyoD-induced promoter activities of MEF2C and MYOG (Myogenin), and reduced histone acetylation near the E-boxes, the MyoD binding site, of the MEF2C and MYOG promoters. Collectively, our results indicate that HDAC11 would suppress myoblast differentiation via regulation of MyoD-dependent transcription. These findings suggest that HDAC11 is a novel critical target for controlling myoblast differentiation.