• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.674-680
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• 2013

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

• 대한화장품학회지
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• 제27권1호
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• 약학회지
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• 제39권2호
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• pp.161-168
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• 1995

Brain dopamine systems play a central role in the control of movement, hormone release, and many complex behavior. The action of dopamine at its synapse is terminated predominately by high affinity reuptake into presynaptic terminals by dopamine transporter (DAT). The dopamine transporter(DAT) is membrane protein localized to dopamine-containing nerve terminals and closely related with cocaine abuse, Parkinsonism, and schizophrenia. In present study, the recombinant plasmid pRc/CMV-DAT, constructed by subcloning of a cDNA encoding a bovine DAT into eukaryotic expression vector pRc/CMV, was stably transfected into CV-1 cells(monkey kidney cell line). The DAT activities in the cell lines selected by Geneticin$^{R}$ were determined by measuring the uptake of $[^3H]$-dopamine. The transfected cell lines showed 30-50 fold higher activities than untransfected CV-1 cell line, and this result implies that DAT is well expressed and localized in transfected cells. The transfected cells accumulated $[^3H]$-dopamine in a dose-dependent manner with a $K_{m}$ of 991.6nM. Even though high doses of norepinephrine, epinephrine, serotonin, and choline neurotransmitters inhibited the uptake of $[^3H]$-dopamine, DAT in transfected cell line was proven to be much more specific to dopamine. The psychotropic drugs such as GBR12909, CFT, normifensine, clomipramine, desipramine, and imipramine inhibited significantly the dopamine uptake in tissue culture cells stably transfected with DAT cDNA. Radioactive in situ hybridization was done to map the cellular localization of DAT mRNA-containing cells in the adult rat central nervous system. The strong hybridization signals were detected only in the substantia nigra pars compacta and ventral tegmental area. The restricted anatomical localization of DAT mRNA-containing cells confirms the DAT as a presynaptic marker of dopamine-containing cells in the rat brain.

• Archives of Pharmacal Research
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• 제24권4호
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• pp.307-311
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• 2001

The activation of NF-$\kappa$B induced by kojic Acid, an inhibitor of tyrosinase for biosynthesis of melanin in melanocytes, was investigated in human transfectant HaCaT and SCC-13 cells. These two keratinocyte cell lines transfected with pNF-$\kappa$B-SEAP-NPT plasmid were used to determine the activation of NF-$\kappa$B. Transfectant cells release the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the NF-$\kappa$B activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selective marker of geneticin resistance. NF-$\kappa$B activation was measured in the SEAP reporter gene assay using a fluorescence detection method. Kojic Acid showed the inhibition of cellular NF-$\kappa$B activity in both human keratinocyte transfectants. It could also downregulate the ultraviolet ray (UVR)-induced activation of NF-$\kappa$B expression in transfectant HaCaT cells. Moreover, the inhibitory activity of kojic Acid in transfectant HaCaT cells was found to be more potent than known antioxidants, e.g., vitamin C and N~acetyl-L-cysteine. These results indicate that kojic Acid is a potential inhibitor of NF-$\kappa$B activation in human keratinocytes, and suggest the hypothesis that NF-$\kappa$B activation may be involved in kojic Acid induced anti-melanogenic effect.

• 한국산림과학회지
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• 제87권2호
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• pp.164-172
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• 1998

Promoter가 없는 외래유전자를 이용하여 형질전환(形質轉換)시키고자 할 때의 최적조건(最適條件) 및 효율적인 형질전환체(形質轉換體)의 검정방법을 구명(究明)하고자 산림청(山林廳) 임목육종연구소(林木育種硏究所)에 식재된 잡종 포플러 양황철 62-9클론을 사용하여 재분화를 유도하고, 기내(器內) 형질전환실험(形質轉換實驗)을 실시하였다. 기내(器內)에서 식물체 재분화 유도를 위해서는 배지조성과 호르몬 농도의 조합이 가장 영향을 많이 끼치는 요인이었다. 여러 조합으로 엽조직 ($5{\times}5mm$ leaf strip)을 시료로 하여 MS 기본배지에 0, 0.01, 0.1, 0.5, $0.1mg/{\ell}$ NAA, 0.2, 0.5, 1.0, $2.0mg/{\ell}$ BAP를 혼합 처리하여 배양한 결과, $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP를 첨가한 호르몬 조합에서 즐기 분화율 및 explant당 분화된 부정줄기수가 상대적인 비율 94%(11.4개)로서 가장 높았다. 따라서 형질전환(形質轉換)된 엽조직(葉組織)을 재분화 시킬 때 이 조건을 줄기분화유도 배지 (SIM ; shoot-inducing medium)로 사용하였다. 선발유전자에 의한 항생제(抗生濟)의 선발농도(選拔濃度)를 알기 위해 pBI121로 형질전환(形質轉換)실험을 한 결과, cocultivation한 후 $100mg/{\ell}$ Km(kanamycin) 또는 $60mg/{\ell}$ G418(geneticin)이 첨가된 SIM 3 배지에서 2주가 경과하면서 육안으로 형질전환(形質轉換)된 부정아를 관찰할 수 있었고 이 부정아를 SIM 3로 옮긴지 6주 후에는 0.5-1cm 크기의 부정줄기로 자랐다. 그러나 대조(對照) 엽조직(葉組織)은 부정아형성이 거의 되지 않아, 선발유전자의 항생제(抗生濟) 선발농도(選拔濃度)는 이 조건이 최적임을 알 수 있었다. 또한 형질전환체(形質轉換體)의 재분화율을 높이기 위해 5-azacytidine을 처리한 결과, 항생제(抗生濟) 선발배지하에서 재분회율을 5.7%에서 26.7%까지 높일 수 있었다. Fluorometric 및 histochemical assay 방법으로 GUS 유전자의 활성(活生)을 검정한 결과 vector system간에 형질전환율(形質轉換率)이 서로 다름을 확인할 수 있었다. LBA4404/pBI121을 vector로 사용한 양황철의 기내(器內) 형질전환(形質轉換) 실험에서는 낮은 形짧훌훌換率(5.7%)을 보였다. 그러나 보다 응양성(應梁性)이 높은 super virulence 유전자를 포함하는 pEHA101을 helper plasmid로 하여 실험한 결과 형질전환율(形質轉換率)이 35.9%로 pAL4404보다 훨씬 효과적인 것으로 나타났다. 따라서 promoter가 없는 외래유전자를 이용하여 promoter tagging을 하고자 할 때 형질전환(形質轉換)실험에는 helper plasmid로써 pEHA101을 이용하는 것이 적합한 것으로 판명되었다.

• 생명과학회지
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• 제16권3호
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• pp.454-458
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• 2006

출아효모인 Sacharomyces cerevisiae S288C균주를 이용한 효모의 게놈이 완성된 후 S. cerevisiae는 다양한 연구 모델로 이용되어져 왔다. 현재까지 효모를 이용한 기능 유전체학 측면에서의 연구는 laboratory strainin인 S288C 균주 또는 그 유래의 균주들이다. 그러나 자연에서 분리된 효모 또는 산업적으로 이용되어지고 있는 S. cerevisiae의 유전학 측면에서의 연구는 낮은 포자형성률 및 형질전환률, 그리고 S288C 균주와의 게놈상의 상이성 때문에 거의 이루어지지 않고 있다. 여기서 우리 연구진은 자연에서 분리된 Saccharomyces cerevisiae KNU5377 균주를 이용하여 random spore analysis를 통해 MATa 및 $MAT{\alpha}$ 타입의 각각의 haploid cell을 분리 후 이미 보고된 KanMX module를 가지고 round PCR기법에 의한 short flanking homology 기법을 이용하여 전사조절인자인 HSF1 유전자가 치환된 변이주를 구축할 수 있었다. 덧붙여, 모든 유전자에 이 기법을 적용할 수는 없다는 것을 확인하였다. 앞으로 이 변이주를 통해 기능 유전체학적인 측면에서 이 유전자의 스트레스와의 관련성을 연구하고자 한다.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.780-803
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• 2003

Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB ($\textrm{NF}_{-{\kappa}}\textrm{B}$) activators [e.g. tumor necrosis factor-$\alpha$, interleukin-1, lipopolysaccharides, and ultraviolet light] leads to phosphorylation and degradation of the inhibitory protein, $\textrm{I}_{{\kappa}}\textrm{B}$. Liberated $\textrm{NF}_{-{\kappa}}\textrm{B}$ is translocated into the nucleus where it can change or alter expression of target genes, resulting in the secretion of extracellular signaling molecules including melanotrophic factors affecting melanocyte. In order to demonstrate the possible role of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of $\textrm{NF}_{-{\kappa}}\textrm{B}$ induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with $\textrm{pNF}_{-{\kappa}}\textrm{B}$-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the $\textrm{NF}_{-{\kappa}}\textrm{B}$ activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then ultraviolet B (UVB) was irradiated. $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the Mis tested, kojic acid ($IC_{50}$/ = 60 $\mu$M) was found to be the most potent inhibitor of UVB-upregulating $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in transfectant HaCaT cells, which is followed by niacinamide ($IC_{50}$/= 540 $\mu$M). Pretreatment of the transfectant HaCaT cells with the Mis, especially kojic acid and niacinamide, effectively lowered $\textrm{NF}_{-{\kappa}}\textrm{B}$ binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion level of IL-6, one of melanotrophic factors, triggered by UV-radiation of the HaCaT cells. These observations suggest that Mis working at the in vivo level might act partially through the modulation of the synthesis of melanotrophic factors in keratinocyte.