• Journal of Life Science
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• v.32 no.5
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• pp.355-361
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• 2022

Melanin pigments are the main cause of skin color. They are produced in melanocytes and then transferred to keratinocytes, which eventually gives the skin surface a variety of colors. Although many skin-lightening or depigmenting agents have been developed, the demand for materials to reduce pig- mentation is still increasing. Here, we tried to find materials for skin-lightening or depigmentation using natural compounds and found that mistletoe (Viscum album var. coloratum) extracts (ME) had an inhibitory effect on tyrosinase activity. As a result, ME significantly reduced pigmentation in human primary melanocytes. In addition, a promoter reporter assay revealed that ME inhibited the transcription of microphthalmia-associated transcription factor (MITF), melanophilin (MLPH), tyrosinase-related protein-2 (TRP-2), and tyrosinase (TYR) genes in HM3KO melanoma cells. In addition, ME decreased the protein level for pigmentation-related molecules, such as TYR and TRP-1. Furthermore, it markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. To elucidate the action mechanism of ME, we investigated its effects on intracellular signaling. Eventually, the ME dramatically decreased the phosphorylation of the cAMP responsive element binding protein (CREB), AKT, and ERK. The data suggest that ME may inhibit the melanogenesis pathway by regulating the signaling pathway related to pigmentation. Taken together, these data propose that ME can be developed as a depigmenting or skin-lightening agent.

• Proceedings of the Korean Society of Toxicology Conference
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• 2000.11a
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• pp.45-67
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• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• Tuberculosis and Respiratory Diseases
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• v.62 no.5
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• pp.406-416
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• 2007

Background: Single nucleotide polymorphisms (SNPs), which consist of a substitution of a single nucleotide pair, are the most abundant form of genetic variations occurring with a frequency of approximately 1 per 1000 base pairs. SNPs by themselves do not cause disease but can predispose humans to disease, modify the extent or severity of the disease or influence the drug response and treatment efficacy. Single nucleotide polymorphisms (SNPs), particularly those within the regulatory regions of the genes often influence the expression levels and can modify the disease. Studies examining the associations between SNP and the disease outcome have provided valuable insight into the disease etiology and potential therapeutic intervention. Traditionally, the genotyping of SNPs has been carried out using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP), which is a low throughput technique not amenable for use in large-scale SNP studies. Recently, TaqMan real-time PCR chemistry was adapted for use in allelic discrimination assays. This study validated the accuracy and utility of real-time PCR technology for SNPs genotyping Methods: The SNPs in promoter sequence (-37 and -524) of lung cancer suppressor gene, RRM1 (ribonucleotide reductase M1 subunit) with the genomic DNA samples of 89 subjects were genotyped using both real-time PCR and PCR-RFLP. Results: The discordance rates were 2.2% (2 mismatches) in -37 and 16.3% (15 mismatches) in -524. Auto-direct sequencing of all the mismatched samples(17 cases) were in accord with the genotypes read by real-time PCR. In addition, 138 genomic DNAs were genotyped using real-time PCR in a duplicate manner (two separated assays). Ninety-eight percent of the samples showed concordance between the two assays. Conclusion: Real-time PCR allelic discrimination assays are amenable to high-throughput genotyping and overcome many of the problematic features associated with PCR-RFLP.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.200-208
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• 2011

Most problematic antibiotic resistance mechanism for MLS (macrolide-lincosamide-streptogramn B) antibiotics encountered in clinical practice is mono- or dimethylation of specific adenine residue at 2058 (E. coli coordinate) of 23S rRNA which is performed by Erm (erythromycin ribosome resistance) protein through which bacterial ribosomes reduce the affinity to the antibiotics and become resistant to them. ErmSF is one of the four gene products produced by Streptomyces fradiae to be resistant to its own antibiotic, tylosin. Unlike other Erm proteins, ErmSF harbors idiosyncratic long N-terminal end region (NTER) 25% of which is comprised of arginine well known to interact with RNA. Furthermore, NTER was found to be important because when it was truncated, most of the enzyme activity was lost. Based on these facts, capability of NTER peptide to inhibit the enzymatic activity of ErmSF was sought. For this, expression system for two different proteins to be expressed in one cell was developed. In this system, two plasmids, pET23b and pACYC184 have unique replication origins to be compatible with each other in a cell. And expression system harboring promoter, ribosome binding site and transcription termination signal is identical but disparate amount of protein could be expressed according to the copy number of each vector, 15 for pACYC and 40 for pET23b. Expression of NTER peptide in pET23b together with ErmSF in pACYC 184 in E. coli successfully gave more amounts of NTER than ErmSF but no inhibitory effects were observed suggesting that there should be dynamicity in interaction between ErmSF and rRNA rather than simple and fixed binding to each other in methylation of 23S rRNA by ErmSF.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.153-158
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• 2008

Tissue-specific and temporal regulation of milk protein gene expression is advantageous when creating transgenic animal that produces foreign protein into milk. Gene expression, i.e. protein production, is regulated not only by promoter strength but also mRNA stability. Especially, poly A tail length by polyadenylation affects in vivo and in vitro mRNA stability and translation efficiency of the target gene. In the present study, nucleotide sequence of 3'-UTR was analyzed to evaluate the effects of mRNA stability on the target gene expression. Based on the poly A signal of 3' -untranslated region (UTR), nucleotide sequences of putative cytoplasmic polyadenylation elements (CPEs) and downstream elements (DSEs: U-rich, G-rich, GU-rich) were analyzed and used to construct 15 luciferase reporter vectors. Each vector was transfected to HC11 and porcine mammary gland cell (PMGC) and measured for dual luciferase expression levels after 48 hours of incubation. Luciferase expression was significantly higher in construct #6 (with CPE 2, 3 and DSE 1 of exon 9) and #11 (with CPE 2, 3 and DSE 1, 2 and 3 of exon 9) than construct #1 in the PMGC. These results suggest that expression of target genes in PMGC may be effectively expressed by using the construct #6 and #11 on production of transgenic pig.