• Title/Summary/Keyword: Functional assay

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Antioxidative and Protective Activity of Polysaccharide Extract from Artemisia iwayomogi Kitamura Stems on UVB-Damaged Mouse Epidermis

  • Ahn, Byung-Yong;Jung, Mun-Yhung
    • Journal of Applied Biological Chemistry
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    • v.54 no.3
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    • pp.184-189
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    • 2011
  • Polysaccharide (PS) was fractionated from hot-water extract of Artemisia iwayomogi Kitamura stems. PS showed considerably higher hydroxyl radical scavenging activity than caffeic acid and glutathione. PS showed lower superoxide anion radical scavenging activity than hydroquinone and ascorbic acid. The scavenging activity of PS on the reactive oxygen species (ROS) induced by human neutrophils with zymosan was determined by the lucigenin-enhanced chemiluminescence assay. The scavenging effect of the PS on ROS as determined by the chemiluminescence assay was about 2-fold stronger than that of ascorbic acid at the same concentration. PS significantly decreased protein carbonyl and malonaldehyde contents in UVB irradiated skin homogenates, which was comparable to glutathione at the same concentration. This result suggested that PS derived from A. iwayomogi Kitamura stems may be a potent candidate as functional compound for the protection on UVB induced skin damage in cosmetics.

The Study of X Chromosome Inactivation Mechanism in Klinefelter's Syndrome by cDNA Microarray Experiment

  • Jeong, Yu-Mi;Chung, In-Hyuk;Park, Jung Hoon;Lee, Sook-Hwan;Chung, Tae-Gyu;Kim, Yong Sung;Kim, Nam-Soon;Yoo, Hyang-Sook;Lee, Suman
    • Genomics & Informatics
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    • v.2 no.1
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    • pp.30-35
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    • 2004
  • To investigate the XIST gene expression and its effect in a Klinefelter's patient, we used Klinefelter's syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter's syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.

Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

  • Kwon, Hye Kyeong;Choi, Hyunwoo;Park, Sung-Gyoo;Park, Woo Jin;Kim, Do Han;Park, Zee-Yong
    • Molecules and Cells
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    • v.44 no.7
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    • pp.500-516
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    • 2021
  • Cardiac hypertrophic signaling cascades resulting in heart failure diseases are mediated by protein phosphorylation. Recent developments in mass spectrometry-based phosphoproteomics have led to the identification of thousands of differentially phosphorylated proteins and their phosphorylation sites. However, functional studies of these differentially phosphorylated proteins have not been conducted in a large-scale or high-throughput manner due to a lack of methods capable of revealing the functional relevance of each phosphorylation site. In this study, an integrated approach combining quantitative phosphoproteomics and cell-based functional screening using phosphorylation competition peptides was developed. A pathological cardiac hypertrophy model, junctate-1 transgenic mice and control mice, were analyzed using label-free quantitative phosphoproteomics to identify differentially phosphorylated proteins and sites. A cell-based functional assay system measuring hypertrophic cell growth of neonatal rat ventricle cardiomyocytes (NRVMs) following phenylephrine treatment was applied, and changes in phosphorylation of individual differentially phosphorylated sites were induced by incorporation of phosphorylation competition peptides conjugated with cell-penetrating peptides. Cell-based functional screening against 18 selected phosphorylation sites identified three phosphorylation sites (Ser-98, Ser-179 of Ldb3, and Ser-1146 of palladin) displaying near-complete inhibition of cardiac hypertrophic growth of NRVMs. Changes in phosphorylation levels of Ser-98 and Ser-179 in Ldb3 were further confirmed in NRVMs and other pathological/physiological hypertrophy models, including transverse aortic constriction and swimming models, using site-specific phospho-antibodies. Our integrated approach can be used to identify functionally important phosphorylation sites among differentially phosphorylated sites, and unlike conventional approaches, it is easily applicable for large-scale and/or high-throughput analyses.

Identification of the Oligotrophic Bacteria Strain 7F Biocontrolling Phytophthora Blight Disease of Red-pepper (고추 역병 방제를 위한 저영양 길항세균 7F 균주의 동정)

  • Kim, Dong-Gwan;Yeo, Yun-Soo;Kwon, Soon-Wo;Jang, Kil-Su;Lee, Chang-Muk;Lee, Mi-Hye;Kim, Soo-Jin;Koo, Bon-Sung;Yoon, Sang-Hong
    • Research in Plant Disease
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    • v.16 no.1
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    • pp.41-47
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    • 2010
  • A total of 10,753 oligotrophic bacteria were isolated from the cultivated soils of red-pepper infected by Phytophthora blight disease in various regions of Korea (Chungju, Anmyon, Taean, Andong, Eumsung and Goesan). Seven bacteria isolates among these collected resources were selected by the first screening of in vitro antagonistic assay against major several plant pathogenic fungi including Phytophthora capsici. Finally, strain 7F was selected by pot assay for a possible biological control agent against Phytophthora blight disease of pepper seedling in the greenhouse. Strain 7F was identified as Bacillus subtilis on the basis of its 16S rDNA sequence analysis and as standardized biochemical characteristics assay kits such as API20 NE. In the experiment of P. capsici zoospore infected red-pepper on the pot test, infection rate of red-pepper with nonetreatment to Phytophthora blight disease was 87%, while the rate was only 6% in the pot treated with strain 7F. This result indicated that the Bacillus subtilis strain 7F will be useful as a potential biocontrol agent for Phytophthora blight disease of red-pepper.

Scutellaria baicalensis Extracts as Natural Inhibitors of Food Browning (천연 갈변저해제로서 황금 추출물의 효소적 갈변 저해 효과)

  • Park, Miji;Chang, Min-Sun;Jeong, Moon-Cheol;Kim, Gun-Hee
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.42 no.5
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    • pp.792-799
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    • 2013
  • This study was designed to develop natural browning inhibitors. The anti-browning effects of distilled water (SBD) and 80% ethanol extracts (SBE) of Scutellaria baicalensis Georgi in apple slices were investigated by L and ${\Delta}E$ values. Both SBD and SBE were effective in reducing the browning of apple slices and were successively fractionated into chloroform ($CHCl_3$), ethyl acetate (EtOAc), and water ($H_2O$) fractions. These extracts were measured for total phenolic content, flavonoid content, anti-oxidative activity (through free radical scavenging activity and the FRAP assay), ferrous ion chelation, and the inhibition of PPO (polyphenol oxidase) activity. Overall, fractions of SBE were better than fractions of SBD in all measurements. The highest total phenolic and flavonoid content were measured in the EtOAc and $CHCl_3$ fractions of SBE. EtOAc and $CHCl_3$ fractions also exhibited the highest anti-oxidative activities (in DPPH and ABTS free radical scavenging and the FRAP assay). Unusually, the highest ferrous ion chelating capacity was found in the $H_2O$ fraction of SBD, but the other fractions showed more than triple the ascorbic acid already in use. Also, $CHCl_3$ fractions showed a stronger inhibition of PPO activity than ascorbic acid. All of these results suggest that EtOAc and $CHCl_3$ fractions from Scutellaria baicalensis can be used as natural anti-browning agents.

THE COMPARISON OF GENE EXPRESSION FROM HUMAN DENTAL PULP CELLS AND PERIODONTAL LIGAMENT CELLS (사람 치수 세포와 치주 인대 세포의 유전자 발현에 관한 비교 연구)

  • Hyoun, So;Park, Sang-Hyuk;Choi, Gi-Woon
    • Restorative Dentistry and Endodontics
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    • v.34 no.5
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    • pp.430-441
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    • 2009
  • The purpose of this study was to characterize functional distinction between human dental pulp cells(PC) and periodontal ligament cells(PDLC) using cDNA micro array assay and to confirm the results of the microarray assay using RT-PCR. 3 genes out of 51 genes which were found to be more expressed(>2 fold) in PC were selected, and 3 genes out of 19 genes which were found to be more expressed(>2 fold) in PDLC were selected for RT-PCR as well. According to this study, the results were as follows: 1. From the micro array assay, 51 genes were more expressed (2 fold) from PC than PDLC. 2. RT-PCR confirmed that ITGA4 and TGF ${\beta}2$ were more expressed in PC than in PDLC 3. From the micro array assay, 19 genes were more expressed (2 fold) from PDLC than PC. 4. RT-PCR confirmed that LUM, WISP1. and MMP1 were more expressed in PDLC than in PC. From the present study, different expression of the genes between the PC and PDLC were characterized to show the genes which play an important role in dentinogenesis were more expressed from PC than PDLC, while the genes which were related with collagen synthesis were more expressed from PDLC than PC.

Isolation and Identification of Two Psoralen Derivatives as Antioxidative Compounds from the Aerial Parts of Angelica keiskei (신선초에 함유된 항산화활성물질 Psoralen 유도체들의 단리 및 동정)

  • Kim, So-Joong;Cho, Jeong-Yong;Wee, Ji-Hyang;Jang, Mi-Young;Rim, Yo-Sup;Kim, Cheol;Shin, Soo-Cheol;Moon, Jae-Hak;Park, Keun-Hyung
    • Korean Journal of Food Science and Technology
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    • v.37 no.4
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    • pp.656-659
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    • 2005
  • Hot water extracts of Angelica keiskei aerial parts were solvent-fractionated with ethyl acetate (EtOAc) and buffers (5% $NaHCO_3$, pH 8.0; 1.0 N HCl, pH 3.0) to obtain EtOAc-soluble acidic and neutral fractions. EtOAc-soluble neutral fraction was purified by Sephadex LH-20 column chromatography and reverse phase HPLC. Assay for purification of antioxidative compounds was performed by spraying DPPH solution on thin layer chromatography. Two isolated substances were identified as pseudoisopsoralen and 8-methoxypsoralen(xanthotoxin) by FAB-MS and NMR analyses.

Aptamers as Functional Nucleic Acids: in vitro Selection and Biotechnological Applications

  • You, Kyung-Man;Lee, Sang-Hyun;Aesul Im;Lee, Sun-Bok
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.2
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    • pp.64-75
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    • 2003
  • Aptamers are functional nucleic acids that can specially bind to proteins, peptides, amino acids. nucleotides, drugs, vitamins and other organic and inorganic compounds. The aptamers are identified from random DNA or RNA libraries by a SELEX (systematic evolution of ligands by exponential amplification) process. As aptamers have the advantage, and potential ability to be released from the limitations of antibodies, they are attractive to a wide range of therapeutic and diagnostic applications. Aptamers, with a high-affinity and specificity, could fulfil molecular the recognition needs of various fields in biotechnology. In this work, we reviewed some aptamer Selection techniques, properties, medical applications of their molecules and their biotechnological applications, such as ELONA (enzyme linked oligonucleotide assay), flow cytometry, biosensors, electrophoresis, chromatography and microarrays.

Functional Characterization of the Major Surface Protein of Treponema maltophilum in Human Gingival Fibroblasts

  • Lee, Sung-Hoon;Choi, Bong-Kyu
    • International Journal of Oral Biology
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    • v.30 no.1
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    • pp.31-37
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    • 2005
  • Treponema maltophilum, a Group IV oral spirochete, is associated with periodontitis and endodontic infections. In this study we analyzed the functional role of the major surface protein of this organism (MspA) in human gingival fibroblasts (HGFs). The full-length gene encoding MspA was cloned and expressed in Escherichia coli by using the expression vector pQE-30. The recombinant protein (rMspA) was purified by affinity chromatography with nickel-nitrilotriacetic acid agarose and possible contamination of E. coli endotoxin in rMspA was removed by using polymyxin B-agarose. rMspA significantly induced the expression of pro inflammatory cytokines like IL-6 and IL-8 and intercellular adhesion molecule (ICAM)-1 in HGFs, when analyzed by reverse transcription-PCR, flow cytometry, and enzyme-linked immunosorbent assay. Our results indicate that MspA of T. maltophilum may play an important role in amplifying the local immune response by upregulating the expression of proinflammatory cytokines and ICAM-1.

Novel CRF1-receptor Antagonists from Pulsatilla koreana Root

  • Li, Wei;Noh, Hyojin;Lee, Sunghou;Lee, Min Ho;Lee, Eun Young;Kang, Sangjin;Kim, Young Ho
    • Natural Product Sciences
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    • v.20 no.4
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    • pp.281-284
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    • 2014
  • In this study, twenty-one oleanane-type triterpenoid saponins were isolated from a methanol extract of the roots of Pulsatilla koreana. Antagonistic activities were measured in these compounds by the aequorin based cellular functional assay system for the corticotropin releasing factor receptor (CRF1). Of them, compounds 7 - 10 showed the highest degree of CRF1 inhibition further at the concentration of $10{\mu}M$. Moreover, by the analysis based on the structure-activity relationship of isolated saponins, a sugar chain at C-3 and a carboxyl group at C-28, as well as a methyl group at C-23 seems to be key functional elements. To our knowledge, this is the first report on CRF1 inhibition of saponins from P. koreana.