• Korean Journal of Microbiology
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• v.49 no.2
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• pp.105-111
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• 2013

The Erm family of adenine-$N^6$ methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Recently, it has been proposed that well conserved amino acids in ErnC' located in concave cleft between N-terminal 'catalytic' domain and C-terminal 'RNA-binding' domain interacts with substrate RNA. We carried out the site-directed mutagenesis and studied the function of the ErmSF R237 mutant in vitro and in vivo. R237 amino acid residue is located in the concave cleft between two domains. Furthermore this residue is very highly conserved in almost all the Erm family. Purified mutant protein exhibited only 51% enzyme activity compared to wild-type. Escherichia coli with R237A mutant protein compared to the wild-type protein expressing E. coli did not show any difference in its MIC (minimal inhibitory concentration) suggesting that even with lowered enzyme activity, mutant protein was able to efficiently methylate 23S rRNA to confer the resistance on E. coli expressing this protein. But this observation strongly suggests that R237 of ErmSF probably interacts with substrate RNA affecting enzyme activity significantly.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.79-86
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• 2012

ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

• Journal of Life Science
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• v.19 no.8
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• pp.1164-1169
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• 2009

Aberrant DNA methylation plays an important role in the development of cancer. It has been reported recently that DNA hypermethylation is involved in the maintenance of cancer stem cells. The present study was designed to test the hypothesis that the demethylating agent, 5-aza-2'-deoxycytidine (AZA), can inhibit the potential for maintenance of cancer stem cells. To validate this hypothesis, we used 4T1 syngeneic mouse models of breast cancer. The AZA pre-treated 4T1 cells showed a dramatic inhibition of tumorsphere formation, compared to their counterparts in vitro. In addition, the AZA treatment significantly suppressed the expression of stem regulator genes, such as oct-4, nanog and sox2, compared to counterparts in vivo. Therefore, selective inhibition of DNA methylation may be useful for stem-specific cancer therapy.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4177-4181
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• 2012

Cancer is a multi-factorial disease and variation in genetic susceptibility, due to inherited differences in the capacity to repair mismatches in the genome, is an important factor in the development of gastric cancer (GC), for example. Epigenetic changes, including aberrant methylation of 5/CpG islands in the promoter regions of mismatch repair (MMR) genes like hMLH1, have been implicated in the development of various types of GC. In the present study we evaluated the role of hMLH1 promoter hypermethylation in Kashmiri GC patients and controls, and assessed correlations with various dietary and lifestyle factors. The study included 70 GC patients (56 males and 14 females; age ($mean{\pm}S.D$) $50{\pm}11.4$ years). Distinction between methylated and unmethylated was achieved with MS-PCR and DNA band patterns. The Chi-square test was applied to assess the risk due to promoter hypermethylation. We found a strikingly high frequency of promoter hypermethylation in GC cases than in normal samples (72.9% (51/70) in GC cases vs 20% (14/70) in normal samples (p=0.0001).We also observed a statistically significant association between methylated hMLH1 gene promoter and smoking, consumption of sundried vegetables and hot salted tea with the risk of GC. This study revealed that hMLH1 hypermethylation is strongly associated with GC and suggested roles for epigenetic changes in stomach cancer causation in the Kashmir valley.

• Genomics & Informatics
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• v.14 no.4
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• pp.187-195
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• 2016

In genetic association studies with high-dimensional genomic data, multiple group testing procedures are often required in order to identify disease/trait-related genes or genetic regions, where multiple genetic sites or variants are located within the same gene or genetic region. However, statistical testing procedures based on an individual test suffer from multiple testing issues such as the control of family-wise error rate and dependent tests. Moreover, detecting only a few of genes associated with a phenotype outcome among tens of thousands of genes is of main interest in genetic association studies. In this reason regularization procedures, where a phenotype outcome regresses on all genomic markers and then regression coefficients are estimated based on a penalized likelihood, have been considered as a good alternative approach to analysis of high-dimensional genomic data. But, selection performance of regularization procedures has been rarely compared with that of statistical group testing procedures. In this article, we performed extensive simulation studies where commonly used group testing procedures such as principal component analysis, Hotelling's $T^2$ test, and permutation test are compared with group lasso (least absolute selection and shrinkage operator) in terms of true positive selection. Also, we applied all methods considered in simulation studies to identify genes associated with ovarian cancer from over 20,000 genetic sites generated from Illumina Infinium HumanMethylation27K Beadchip. We found a big discrepancy of selected genes between multiple group testing procedures and group lasso.

• Environmental Mutagens and Carcinogens
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• v.18 no.2
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• pp.77-82
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• 1998

In order to compare the suppressive effect of galangin on the genotoxicity by N-methyl-N-nitrosourea (MNU) or benzo[a]pyrene B(a)P, in vivo micronycleus test using mouse peripheral blood and in vitro sister chromatid exchange(SCE) test using mouse spleen lymphocytes were performed. MNU or B(a)P-induced micronucleated reticulocytes in vivo was decreased by the simultaneous treatment of galangin. MNU or B(a)P-induced SCEs in vitro was also decreased by the simultaneous treatment of galangin. On the other hand, the determinations of [$^3$H]MNU-induced total DNA binding and methylated DNA were performed to find out the mechanism of action. [$^3$H]MNU-induced total DNA binding was inhibited by the treatment of galangin in calf thymus DNA. HPLC analysis of DNA hydrolysates showed that galangin caused a decrease of 7-methyl guanine and $O^{6}$-methyl guanine in calf thymus DNA. To elucidate the action mechanism of galangin against B(a)P, alteration of B(a)P metabolism was studied. Galangin inhibited B(a)P metabolism in the presence of S-9 mix and decreased B(a)P-DNA binding in calf thymus DNA with S-9 mix.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2145-2150
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• 2015

HPV viruses are integral to the development of cervical cancer. The pathogenesis has been extensively studied. To date, numerous HPV tests and products have been developed and successfully utilized in diagnosis, treatment and prevention of cervical cancer. The HPV DNA test, when combined with other routine cervical cancer screening and diagnostic tests namely exfoliative cytology, visual inspection with acetic acid (VIA) and colposcopy has increased the detection rate of cervical cancer. HPV DNA products could also be measured in other body fluids like urine, lymph node tissue, and serum. HPV association could also be quantified by measuring other parameters like HPV mRNA, viral load, viral integration and methylation status. Vaccination against HPV has been found to decrease the incidence of cervical cancer. Further, therapeutic vaccines for cervical cancer against HPV continue to evolve. All these findings pertaining to HPV could possibly decrease the incidence of cervical cancer in the near future. This review aims to give an overview of the HPV tests and products in use and those under trial currently.

• Biomedical Science Letters
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• v.11 no.2
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• pp.165-173
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• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• YAKHAK HOEJI
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• v.35 no.4
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• pp.341-347
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• 1991

It was reported that protein carboxymethylation is involved in amylase secretion of parotid gland by isoproterenot. It was also suggested that a small part of the total cellular protein carboxymethylation is directly involved in pancreatic enzyme secretion. On the contrary, other authors reported that there is no relationship between protein carboxymethylation and secretion in pancreas and parotid gland. In recent study, it was proposed that a methyl acceptor protein plays a limited modulatory role in the coupling of cytosolic $Ca^{++}$ accumulation and exocytosis. In this study, the effects of cholinergic and adrenergic agents on the activities of protein methylase II in pancreatic tissues were examined to test the relationship between protein methylation and pancreatic secretion. The results are as follows. The activity of amylase was slightly increased at the concentration of $10^{-5}$ M of isoproterenol and norepinephrine. The activities of protein methylase I and II were decreased by isoproterenol and norepinephrine, but the activities of protein methylase III were hardly changed. The cholinergic stimulants acetylcholine and carbachol at a concentration of $10^{-5}$ M increased the activities of protein methylase I and decreased the activitiy of protein methylase III compared with control.