• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3817-3825
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• 2015

Background: The human protein methyl-transferase DOT1L catalyzes the methylation of histone H3 on lysine 79 (H3K79) at homeobox genes and is also involved in a number of significant processes ranging from gene expression to DNA-damage response and cell cycle progression. Inhibition of DOT1L activity by shRNA or small-molecule inhibitors has been established to prevent proliferation of various MLL-rearranged leukemia cells in vitro, establishing DOT1L an attractive therapeutic target for mixed lineage leukemia (MLL). Most of the drugs currently in use for the MLL treatment are reported to have low efficacy, hence this study focused on various natural compounds which exhibit minimal toxic effects and high efficacy for the target receptor. Materials and Methods: Structures of human protein methyl-transferase DOT1L and natural compound databases were downloaded from various sources. Virtual screening, molecular docking, dynamics simulation and drug likeness studies were performed for those natural compounds to evaluate and analyze their anti-cancer activity. Results: The top five screened compounds possessing good binding affinity were identified as potential high affinity inhibitors against DOT1L's active site. The top ranking molecule amongst the screened ligands had a Glide g-score of -10.940 kcal/mol and Glide e-model score of -86.011 with 5 hydrogen bonds and 12 hydrophobic contacts. This ligand's behaviour also showed consistency during the simulation of protein-ligand complex for 20000 ps, which is indicative of its stability in the receptor pocket. Conclusions: The ligand obtained out of this screening study can be considered as a potential inhibitor for DOT1L and further can be treated as a lead for the drug designing pipeline.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.771-776
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• 2001

A pregnant-induced clone was identified by differential screening from a cDNA library of bovine mammary gland. The clone was identified as a cDNA encoding a polyadenylate binding protein 1 (PABP). The cDNA clone had a total length of 1,911 nucleotides coding for 636 amino acids. The nucleotide sequence of the bovine PABP was 95% and 94% identical to those of human and mouse species, respectively. Comparison of the deduced amino acid sequences of bovine PABP with those of human species showed 100% identity. Induction of the PABP mRNA was observed in bovine mammary tissues at pregnant 7 and 8 months compared to virgin, lactating and involuted states. Expression of the PABP gene was examined in mammary epithelial HC11 cells at proliferating, differentiated and apoptotic conditions. The mRNA levels of PABP gene were similar between proliferating and differentiated cells, but expression levels were very low in apoptotic cells compared to other conditions. Results demonstrate that the PABP gene is induced during pregnancy at which stage mammary epithelial cells are actively proliferating.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7091-7094
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• 2013

DNA repair capacity is crucial in maintaining cellular functions and homeostasis. However, it can be altered based on DNA sequence variations in DNA repair genes and this may lead to the development of many diseases including malignancies. Identification of genetic polymorphisms responsible for reduced DNA repair capacity is necessary for better prevention. Homologous recombination (HR), a major double strand break repair pathway, plays a critical role in maintaining the genome stability. The present study was performed to determine the frequency of the HR gene XRCC3 Exon 7 (C18067T, rs861539) polymorphisms in Saudi Arabian population in comparison with epidemiological studies by "MEDLINE" search to equate with global populations. The variant allelic (T) frequency of XRCC3 (C>T) was found to be 39%. Our results suggest that frequency of XRCC3 (C>T) DNA repair gene exhibits distinctive patterns compared with the Saudi Arabian population and this might be attributed to ethnic variation. The present findings may help in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1683-1686
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• 2012

In the present case control study mRNA expression of the GSTP1 gene, encoding a phase II enzyme that detoxifies via glutathione conjugation, was investigated using semiquantitative PCR followed by SSCP for 49 confirmed head and neck (HN) cancer and 49 control samples. It was found that GSTP1 was upregulated in significantly higher number of cancers (OR 4.2, 95% CI 1.2-15.3). Grade wise correlation was also observed with more up regulation in patients with more advanced grades of HN carcinomas. We also found that 5 patients showed variation in mRNA with a larger product size than expected. Sequencing revealed insertion of an intronic segment between the $6^{th}$ and $7^{th}$ exon of the GSTP1 gene. Germline screening was performed showing mobility shifts which suggested mutation at the DNA level resulting in intronic portion retention. This study is of prime importance for drug design and treatment selection to overcome increased resistance of HN cancers to drugs due to alteration in the GSTP1 gene.

• Toxicological Research
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• v.17 no.1
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• pp.65-71
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• 2001

The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. A yeast-based steroid hormone receptor gene trascription assay was previously developed for the evaluation of chemicals with endocrine modulating activity. The yeast transformants used in this assay contain the human estrogen receptor along with the appropriate steroid response elements upstream of the $\beta$-galactosidase reporter gene. We tried to evaluate several natural and synthetic steroids of their potential to interact directly with the steroid receptor. Some putative endocrine disruptors, including nonylphenol, are weakly estrogenic. But the combined treatment oj these chemicals with di-(2-ethylhexyl)phthalate (DEHP) significantly increased the $\beta$-galactosidase activity in the yeast transformant. These results suggest that we also have to consider the synergistic effects of endocrine disruptors. In this study, we showed that yeast-based bioassay is a valuable tool for screening potential endocrine disruptors and quantitative determination of estrogenicity. And the possibility that the estrogen receptor binds multiple environmental chemicals adds another level of complexity to the interaction between the endocrine disruptors and the human hormone system.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.1725-1727
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• 2016

Breast cancer contributes to approximately 23% of the cancer cases identified and 14% of cancer related deaths worldwide. Including a strong association between genetic and environmental factors, breast cancer is a complex and multi factorial disorder. Two high penetration breast cancer susceptibility genes (BRCA1 and BRCA2) have been identified, and germ line mutations in these are thought to account for between 5% and 10% of all breast cancer cases. The human BRCA1 gene, located on 17q, is involved in the regulation of cell proliferation by aiding in DNA repair, transcriptional responses to DNA damage and cell cycle check points. Mutations in this gene enhance cell proliferation and facilitate formation of tumors. Two mutations, the 185 deletion of AG and the 4627 substitution from C to A, are founder mutations in the BRCA1 gene for breast cancer in Asian populations. Allele specific PCR was performed to detect these selected mutations in 120 samples. No mutation of 4627 C to A was detected in the samples and only one of the patients had the 185 del AG mutation in the heterozygous condition. Our collected samples had lower consanguinity and family history indicating the greater involvement of environmental as compared to genetic factors.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.316-319
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• 1988

Pectate Lyase (PL) was cloned from alkali-tolerant Bacillus sp. YA-14 into Escherichia coli MB1000 by inserting HindIII-generated DNA fragment into the HindIII site of pBR322 and then screening recombinant transformant for the ability to hydrolyze sodium polypectate on agar plate, The recombinant plasmid, called pYPC29, was isolated, and the size of the cloned HindIII fragment was found to be 1.6 kb. The PL gene was stablely maintained and expressed efficiently in Escherichia coli. The Pt accumulated largely in the periplasmic space of Escherichia coli clones.

• Journal of Acupuncture Research
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• v.20 no.3
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• pp.34-44
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• 2003

Objective : Bone homeostasis is maintained by balance of bone formation and resorption. Therefore, bone related diseases arose by disturbance of this balance between osteoblast and osteoclast activities. To develop a successful screening system the therapeutic components based on oriental medicine is essential to set up systematic approach for that purpose. The purpose of this study is to the know the gene expression using cDNA microarray assay. Methods : Cervi Pantotricuhum Cornu Herbal-acupuncture extract was prepared by boiling. human osteosarcoma cells(HOS) were treated with Cervi Pantotricuhum Cornu Herbal-acupuncture solution. Then mRNA was extracted and cDNA microarray assay was performed. Results : Human osteosarcoma cells(HOS) treated with Cervi Pantotricuhum Cornu Herbal-acupuncture solution($500{\mu}g/m{\ell}$) showed that thioredoxin, TAFII31 and two novel genes were increased. However many genes decreased their expression by Cervi Pantotricuhum Cornu Herbal-acupuncture. Conclusions : This type of approach will give a good chance to explore the favorable effects of Cervi Pantotricuhum Cornu Herbal-acupuncture. Further study is needed for investigating the effect of Cervi Pantotricuhum Cornu Herbal-acupuncture.