11:10-12:00, Rm 103

OthersU-18-01Use of non-melanocytic HEK293 cells stably expressing humantyrosinase for the screening of anti-melanogenic agentsMijin Kim and Yong Chool BooDepartment of Molecular Medicine and Cell and Matrix Research Institute, BK21 MedicalEducation Program for Human Resources, Kyungpook National University School of Medicine,Daegu 700-422, KoreaTyrosinase (TYR) from mushrooms has been inappropriately used in screening assay forhypo-pigmenting agents even though its biochemical properties are different from thoseof human TYR. Cell-free extracts of human epidermal melanocyes (HEMs) could beanother choice for the assay but HEMs grow too slowly to get a sufficient amount of cellfree extracts. In the present study, human embryonic kidney (HEK) 293 cells weretransfected with a human TYR construct to establish a cell line that grows rapidly andexpresses human TYR constitutively. Cell-free extracts of the established cell line,HEK293-TYR, were tentatively used in the screening assays for variousphenylpropanoids that have chemical structures similar to L-tyrosine, the substrate ofTYR. Among the 11 compounds, the strongest inhibition of TYR activity was shown by p-coumaric acid (IC50, 3 μM), followed by 3-(4-hydroxyphenyl)propionic acid (50 μM) and 3-(4-hydroxyphenyl)lactic acid (70 μM). The effects of these phenylpropanoids on themelanin synthesis in HEMs correlated well with their effects on TYR activity in vitro. Thisstudy demonstrated that HEK293-TYR cells can be a good source of human TYRenzyme needed in screening assay of anti-melanogenic agents.U-18-04Comparison of the antimelanogenic effects of p-coumaric acid and itsmethyl ester in human epidermal melanocytes and their skinpermeabilities ex vivoKyosun Song and Yong Chool BooDepartment of Molecular and Cell and Matrix Reasearch Institute, BK21 Medical EducationProgram for Human Resources, Kyungpook National University School of Medicine, Daegu 700-422, Koreap-Coumaric acid (PCA) inhibits human tyrosinase (TYR) activity and melanin synthesis inhuman epidermal melanocytes. The purpose of the current study was to further explorePCA’s potential as a hypopigmenting agent for cosmetic use. PCA and methyl p-coumarate (MPC) were comparatively tested against in vitro human TYR enzyme activityand cellular melanin synthesis in human epidermal melanocytes. Permeation studieswere undertaken using an artificial lipophilic membrane and an excised porcine skin.Although PCA was a stronger inhibitor than MPC against TYR activity in vitro, the formerinhibited cellular melanin synthesis less effectively than the latter. A non-cell basedpermeability assay indicated that PCA was practically impermeable through the lipophilicbarrier while MPC was highly permeable. In contrast, an ex vivo skin permeation studydemonstrated that topically applied PCA in the form of a cosmetic cream can diffuse intothe aqueous medium underneath the skin. No MPC was released from a MPC cream butPCA was released instead as a bio-converted product. PCA may be useful for topicalapplications for a hypo-pigmenting effect. MPC may also have great potential as ahypopigmenting agent but requires rather invasive methods for its delivery to the targetcells.U-18-02Regulatory mechanisms for endothelial argininosuccinate synthetase 1expression by laminar shear stress and cellular senescenceGyeong In Mun and Yong Chool BooDepartment of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical EducationProgram for Human Resources, Kyungpook National University School of Medicine, Daegu 700-422, KoreaEndothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of L-arginineto nitric oxide synthase 3 (NOS3) that produces nitric oxide at the expense of L-arginine.The purpose of the present study was to elucidate the mechanisms responsible for thedifferential expression of ASS1 in young and senescent endothelial cells (ECs) understatic and laminar shear stress (LSS) conditions. Analysis of transcriptomes by a cDNAmicroarraydemonstrated that expression of many Kruppel-like factors (KLFs) includingKLF2 and KLF4 were up-regulated by LSS both in young and senescent HUVECs.Essential roles of KLF2 and KLF4 in the LSS-induced expression of ASS1 wereconfirmed the depletion of KLF2 and KLF4 by pre-treating young cells with siRNAs ofKLF2 and KLF4. The in silico analysis of ASS1 promoter region using CpG plot softwareidentified two putative CpG islands comprising the regions between -217/+56 and+278/+764. Treatment of senescent ECs with 5’-aza-2’-deoxycytidine led to a significantincrease of ASS1 expression, indicating ASS1 expression might be suppressed insenescent ECs by promoter methylation. The present study demonstrated that bothgenetic acid epigenetic mechanisms are involved in the regulation of ASS1 expression inresponse to LSS and cellular senescence.U-18-05Expression of the Mup2 gene is controlled by two regulators, circadianclock and glucocorticoid signalYun-Hee Cho, Young-Su Jang, Young-Ro Lee and Kiho Bae*Division of Biological Science and Technology, Yonsei University, Wonju 220-710, KoreaIn mammals, the suprachiasmatic nuclei (SCN) in the brain function as the masterregulators of circadian rhythm and coordinate the oscillators in peripheral organs. Lossesof clock genes alter gene expression and behavior and there are interactions between thecircadian clock and the hypothalamic-pituitary-adrenal axis. Based on these findings, weinvestigated whether disruption of the circadian clock and glucocorticoid signals wouldinfluence the gene expression of major urinary protein (Mup) in the liver. Both Mup2mRNA and protein showed biphasic rhythms with similar phase, irrespective of genotype.However, the peak of the rhythm is shifted in mPeriod2 (mPer2) mutant mice at amaximum of 8 hours. We identified two E boxes and one glucocorticoid responseelement (GRE) as regulatory elements for Mup2 expression via reporter assay. WhileCLOCK binds to the E-boxes constantly, GR was capable of binding to the GRE in atime-of-day specific manner, binding more strongly at ZT1 in wild-type than in the mPer2mutant mice, which is consistent with its protein levels. Taken together, our resultsindicate that Mup2 expression is tightly regulated by both the circadian clock andglucocorticoid.U-18-03Kinetic studies of pig liver aldehyde dehydrogenaseYong-Kweon Cho and Hyun-Woo SimDepartment of Biochemistry and Health Science, Changwon National University, Changwon,Gyeongnam 641-939, KoreaTo determine the kinetic mechanism of pig liver liver aldehyde dehydrogenase, initialvelocity studies were carried out. The data showed that the enzyme reaction followssequential mechanism, where substrates bind prior to release of products. Data fromdead-end inhibition studies with thionocotinamide-AD+, N’-methylnicotinamide, adenine,adenosine and NADH showed that NAD+ and aldehyde can bind in random fashion. Theresults from product inhibition of NADH and glyceraldehyde supported the abovemechanism.U-18-06Real-time RT-PCR method for detection of breast cancer diagnosticmarkerYoonjung Cho¹, Jaewon Lim¹, Sangjung Park¹, Dongsup Lee¹ , ², Kwanghwa Park²,In Soo Lee³, Hyeyoung Lee¹and Tae-Ue Kim¹¹Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University,Wonju 220-710, ²Department of Pathology, College of Medicine, Yonsei University WonjuChristian Hospital, Wonju 220-701, ³Department of Clinical Laboratory Science, Hyejeon College,Hongseong 350-702, KoreaBreast cancer is the most frequently diagnosed cancer in female population of the world,and the morbidity of this malignant disease is increasing every year. Earlier detection ofbreast cancer leads to better prognosis and lower mortality. ER (estrogen receptor), PR(progesterone receptor) and HER2 (human epidermal growth factor receptor 2) are veryimportant predictive and prognostic markers for breast cancer. Currently determination ofER, PR and HER2 overexpression in breast cancer tissues is routinely performed by(IHC) immunohistochemistry or FISH (fluorescence in situ hybridization analysis).However these tests have several limitations. We developed a real-time RT-PCR (realtimereverse transcription polymerase chain reaction) assay to quantify the ER, PR andHER2 expression with FFPE (formalin-fixed paraffin-embedded) tissue samples frombreast cancer. Preliminary results suggest that ER, PR and HER2 expressionapproximately 10-500 times higher than negative tissue samples. Real-time RT-PCRprovides significantly correlated data in FFPE tissue samples. Our method is rapid,sensitive and cost-effective. Finally this method could be used to identify breast cancerpatients with poor prognosis and to suggest determination of chemotherapeutic method.340 Korean Society for Biochemistry and Molecular Biology