• Journal of Life Science
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• v.30 no.2
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• pp.113-121
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• 2020

Macrophages are initiators for regulating a host's defenses to eliminate pathogens and trigger tissue repair. Macrophages are classified into two types: classically (M1) activated macrophages and alternatively (M2) activated macrophages. M1-phenotype macrophages directly or indirectly kill infectious organisms and tumor cells via pro-inflammatory responses, whereas M2-phenotype macrophages remodel wounded tissue through anti-inflammatory responses. In this paper, we investigated how Phellinus linteus hot water extract passed from Diaion HP-20 resin (PLEP) regulates polarization of M1-like or M2-like macrophages in human THP-1 cells. PLEP did not have cytotoxicity at a high concentration of 300 ㎍/ml. We observed morphological alteration of the THP-1 cells, which are stimulated by PLEP, LPS/INF-γ (M1 stimulators) or IL-4/IL13 (M2 stimulators). PLEP exposure induced morphology contiguous with LPS/INF-γ. qPCR was also performed to determine whether PLEP influences M1 or M2 polarization-related genes. M1-phenotype macrophage-specific genes, such as TNF-α, IL-1β, IL-6, IL-8, CXCL10 and CCR7, were enhanced by PLEP in a dose-dependent manner similar to LPS/INF-γ. Conversely, M2-phenotype-specific genes, such as MRC-1, DC-SIGN, CCL17 and CCL22, were suppressed by PLEP. PLEP also significantly up-regulated secretory inflammation cytokines related to M1 polarization of macrophages, including TNFα, IL-1β and IL-6, which was similar to the gene expression. Further, MAPK and NF-κB signaling were increased by treatment with PLEP, resulting in enhancement of cytokine secretion. PLEP might therefore be used as a promising booster of pro-inflammatory responses through M1 polarization of human THP-1 cells.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.41 no.4
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• pp.325-331
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• 2015

Skin is exposed to sunlight or artificial indoor light on a daily. The reached solar light on the earth surface consist of 50% visible light and 45% infrared (IR) except for ultra violet (UV). The negative effects of UV including UVB and UVA have been steadily investigated within the last decades. However, little is known about the effects of visible or IR light. In this study, we irradiated human dermal fibroblasts using light emitting diode (LED) to investigate the optimal parameter for enhancing cell growth and collagen synthesis. We found that red of 630 nm and green of 520 nm enhance the cell proliferation, but irradiation with purple and blue light exerts toxic effects. To examine the response of irradiation time and light intensity on the fibroblasts, cells were exposed to red or green light with intensities from 0.05 to $0.75mW/cm^2$. Procollagen secretion was increased of 1.4 fold by 10 min irradiation, while 30 min treatment decreased the collagen synthesis of dermal fibroblasts. Treatment with red of $0.3mW/cm^2$ and green of 0.15 and $0.3mW/cm^2$ resulted in enhancement of collagen mRNA. Lastly, we investigated the combinatorial effect of red and green light on dermal fibroblasts. The sequential irradiation of red and green light is an efficient way for the purpose of the increase in the number of fibroblasts than single light treatment. On the other hand, the exposure of red light alone was more effective method for enhancing of collagen secretion. Our study showed that specific light parameters accelerated cell proliferation, gene expression and collagen secretion on human dermal fibroblasts. In conclusion, we demonstrate that light exposure with specific parameter has beneficial effects on the function of dermal fibroblasts, and suggests the possibility of its cosmetically and clinical application.

• Journal of Bio-Environment Control
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• v.32 no.1
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• pp.23-33
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• 2023

Sucrose (suc) is a disaccharide that consists of glucose (glu) and fructose (fru). It is a carbohydrate source that acts as a nutrient molecule and a molecular signal that regulates gene expression and alters metabolites. This study aimed to evaluate whether suc-specific signaling induces an increase in bioactive compounds by exogenous suc absorption via roots or whether other factors, such as osmotic stress or biotic stress, are involved. To compare the osmotic stress induced by suc treatment, 4-week-old cultured mugwort plants were subjected to Hoagland nutrient solution with 10 mM, 30 mM, and 50 mM of suc or mannitol (man) for 3 days. Shoot fresh weight in suc and man treatments was not significantly different from the control. Both man and suc treatments increased the content of bioactive compounds in mugwort, but they displayed different enhancement patterns compared to the suc treatments. Mugwort extract treated with suc 50 mM effectively protected HepG2 liver cells damaged by ethanol and t-BHP. To compare the biotic stress induced by suc treatment, 3-week-old mugwort plants were subjected to microorganism and/or suc 30 mM with Hoagland nutrient solution. Microorganisms and/or suc 30 mM treatments showed no difference about the shoot fresh weight. However, sugar content in mugwort treated with suc 30 mM and microorganism with suc 30 mM treatment was significantly higher than that of the control. Suc 30 mM and microorganism with suc 30 mM were effective in enhancing bioactive compounds than microorganism treatment. These results suggest that mugwort plants can absorb exogenous suc via roots and the enhancement of bioactive compounds by suc treatment may result not from osmotic stress or biotic stress because of microorganism, but by suc-specific signaling.

• Journal of Life Science
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• v.28 no.7
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• pp.835-840
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• 2018

Quantitative determination of the protein expression levels is one of the most important parts in assessment of the safety of foods derived from genetically modified (GM) crops. Overexpression of AtCYP78A7, a gene encoding cytochrome P450 protein, has been reported to improve tolerance to abiotic stress, such as drought and salt stress, in transgenic rice (Oryza sativa L.). In the present study, an enzyme-linked immunosorbent assay (ELISA) kit for diagnosing AtCYP78A7 protein including AtCYP78A7-specific monoclonal antibody was developed. GST-AtCYP78A7 recombinant protein was induced and purified by affinity column. Four monoclonal antibodies (mAb 6A7, mAb 4C2, mAb 11H6, and mAb 7E8) against recombinant protein were also produced and biotinylated with avidin-HRP. After pairing test using GST-AtCYP78A7 protein and lysate of rice samples, mAb 4C2 and mAb 7E8 were selected as a capture antibody and a detecting antibody, respectively, for ELISA kit. Product test using rice samples indicated that percentages of detected protein in total protein were greater than 0.1% in AtCYP78A7-overexpressing transgenic rice (Line 10B-5 and 18A-4), whereas those in negative control non-transgenic rice (Ilpum and Hwayoung) were less than 0.1%. The ELISA kit developed in this study can be useful for the rapid detection and safety assessment of transgenic rice overexpressing AtCYP78A7.

• Journal of Life Science
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• v.24 no.11
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• pp.1157-1167
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• 2014

In order to compare the anti-inflammatory effects of five selected cereal grains-proso millet, hwanggeumchal sorghum, foxtail millet, barnyard millet, and adlay-the inhibitory activities of 80% ethanol (EtOH) extracts obtained from the individual grains on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation were investigated in RAW264.7 cells. The EtOH extract of barnyard millet (Echinochloa crus-galli var. frumentacea) grains exhibited more potent anti-inflammatory activity than that of the other grains. When the EtOH extract of barnyard millet grains was sequentially fractionated with n-hexane, methylene chloride (MC), ethyl acetate (EtOAc), and n-butanol, the majority of the anti-inflammatory activity was detected in the MC fraction, followed by the EtOAc fraction. Pretreatment with the MC fraction caused downregulation of the expression levels of iNOS- and COX-2-specific transcripts and proteins, as well as proinflammatory cytokine gene transcripts (IL-$1{\beta}$, IL-6, and TNF-${\alpha}$) in LPS-stimulated RAW264.7 cells. Additionally, the MC fraction could suppress not only the LPS-induced nuclear translocation of cytosolic NF-kB, but also the LPS-induced activation of MAPKs, such as ERK, JNK, and p38MAPK. Further analysis of the MC fraction by HPLC identified kaempferol, biochanin A, and formononetin as the major phenolic components. Both kaempferol and biochanin A, but not formononetin, could exert anti-inflammatory effect at the same concentrations as those of the MC fraction. Consequently, these results indicate that kaempferol and biochanin A are among the most effective anti-inflammatory phenolic components in barnyard millet grains. This finding suggests that barnyard millet grains and the MC extract enriched in kaempferol and biochanin A could be beneficial functional food sources that have an anti-inflammatory effect.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.455-462
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• 2016

Pseudomonas aeruginosa P-5 is an unusual organism capable of synthesizing polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyvalerate (3HV) and medium-chain-length (MCL) 3-hydroxyalkanoate (3HA) monomer units when C-odd alkanoic acids are fed as the sole carbon source. Evaluation of the substrate chain-length specificity of two P. aeruginosa P-5 PHA synthases ($PhaC1_{P-5}$ and $PhaC2_{P-5}$) by heterologous expression of $PhaC1_{P-5}$ and $PhaC2_{P-5}$ genes in Pseudomonas putida GPp104 revealed that $PhaC2_{P-5}$ incorporates both 3HV and MCL 3HAs into PHA, whereas $PhaC1_{P-5}$ favors only MCL 3HAs for polymerization. In order to obtain $PhaC2_{P-5}$ mutants with altered substrate specificity, site-specific mutagenesis for $PhaC2_{P-5}$ was conducted. Amino acid substitutions of $PhaC2_{P-5}$ at two positions (Ser326Thr and Gln482Lys) were very effective for synthesizing copolymers with a higher 3HV fraction. When recombinant P. putida GPp104 harboring double mutated $phaC2_{P-5}$ gene ($phaC2_{P-5}QKST$) was grown on nonanoic acid, 2.5-fold increase of copolymer content with 3.8-fold increase of 3HV fraction was observed. The $phaC2_{P-5}QKST$-containing Ralstonia eutropha PHB-4 supplemented with valeric acid also produced copolymers consisting of 3HV and 3-hydroxyheptanoate with a high 3HV fraction. These results suggest that recombinants containing $phaC2_{P-5}QKST$ could be useful for production of new PHA copolymers with improved material properties.

• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.487-494
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• 2008

Background: Chromosome 17p allele losses and mutations of p53 gene are the most common genetic abnormalities in lung cancer. The purposes of this study were to evaluate the factors associated with p53 protein overexpression and to evaluate its prognostic value in patients with pathologic stage I non-small cell lung cancer (NSCLC). Methods: This is a retrospective review for the patients who underwent surgical resection at Samsung Medical Center between Jan 2003 and Jun 2004. Immunohistochemical staining for p53 protein was performed on tumor tissues from patients with lung cancer. The p53 overexpression was evaluated in relation to age, sex, smoking history, histology and pathologic stage by univariate and multivariate analyses. The disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS) were analyzed using the Kaplan-Meier methods and the differences in DFS, DSS and OS were assessed by using the log-rank tests. Results: A total of 125 patients were included in the analysis and a median frequency of p53 expression in tumor tissue was 10%. The p53 overexpression (${\geq}10%$) was more common in squamous cell carcinoma (66%) than in adenocarcinoma (38%, p=0.002). The p53 overexpression was more common in pathologic stage IB (59%) than in IA (38%, p=0.002). Patients with p53-overexpressing tumor (27 years) smoked more years compared with those without it (20 years, p=0.032). Smoking history ${\geq}25$ pack-years was more common in patients with p53 overexpression (58%) than in those without it (38%, p=0.024). In the multivariate analysis, only histology was significantly associated with p53 overexpression. However, there were no significant differences of DFS, DSS and OS in relation to p53 status. Conclusion: The p53 overexpression was associated with histology, pathologic stage and smoking history in patients with pathologic stage I NSCLC. However, the p53 overexpression was not associated with patient's survival.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.3
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• pp.470-486
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• 1995

Mammary epithelial cells contain a subpopulation of cells with a large proliferativ potential which are responsible for the maintenance of glandular cellularity and are the progenitor cells of mammary cancer. These clonogens give rise to multicellular clonal alveolar or ductal units(AU or DU) on transplantation and hormonal stimulation. To isolate putative mammary clonogens, enzymatically monodispersed rat mammary epithelial cells from organoid cultures and from intact glands are sorted by flow cytometry according to their affinity for FITC labeled peanut lectin(PNA) and PE labeled anti-Thy-1.1 antibody(Thy-1.1) into four subpopulations : cells negative to both PNA and Thy-1.1(B-), PNA+cells, Thy-1.1+cells, and cells positive to both reagents(B+). The in vivo transplantation assays indicate that the clonogenic fractions of PNA+cells from out-growths of organoids in primary cultures for three days in complete hormone medium(CHM) are significantly higher than those of cells from other subpopulations derived from cultrues or from intact glands. Extracellular matrix(ECM) is a complex of several proteins that regulated cell function ; its role in cell growth and differentiation and tissue-specific gene expression. It can act as a positive as well as a negative regulator of cellular differentiation depending on the cell type and the genes studied. Regulation by ECM is closely interrelated with the action of other regulators of cellular function, such as growth factors and hormones. Matrigel supports the growth and development of several different multicellular colonies from mammary organoids and from monodispersed epithelial cells in culture. Several types of colonies are observed including stellate colonies, duct-like structures, two- and three-dimensional web structures, squamous organoids, and lobulo-duct colonies. Organoids have the greatest proliferative potential and formation of multi-cellular structures. Phase contrast micrographs demonstrate extensive intracellular lipid accumulation within the web structures and some of duct-like colonies. At the immunocytochemical and electron micrograph level, casein proteins are predominantly localized near the apical surface of the cells or in the lumen of duct-like or lobulo-duct colonies. Squamous colonies are comprised of several layers of squamous epithelium surrounding keratin pearls as is typical fo squamous metaplasia(SM). All-trans retinoic acid(RA) inhibits the growth of SM. The frequency of lobulo-ductal colony formation increased with the augmentation of RA concentration in these culture conditions. The current study models could provide powerful tools not only for understanding cell growth and differentiation of epithelial cells, but also for the isolation and characterization of mammary clonogenic stem cells.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.