• Tuberculosis and Respiratory Diseases
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• 제70권1호
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• pp.21-27
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• 2011

Background: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of $EGFR$ mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to $EGFR$ mutation status using both methodologies. Methods: Clinical specimens from 112 NSCLC patients were analyzed for $EGFR$ mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to $EGFR$-TKIs were compared. Results: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). Conclusion: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in $EGFR$ gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of $EGFR$ mutations in clinical setting.

• 한국초지조사료학회지
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• 제34권4호
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• pp.262-268
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• 2014

In order to reveal the aluminum (Al) stress tolerance mechanisms in alfalfa plant at low pH soil, a proteomic approach has been conducted. Alfalfa plants were exposed to Al stress for 5 days. The plant growth and total chlorophyll content are greatly affected by Al stress. The malondialdehyde (MDA) and $H_2O_2$ contents were increased in a low amount but free proline and soluble sugar contents, and the DPPH-radical scavenging activity were highly increased. These results indicate that antioxidant activity (DPPH activity) and osmoprotectants (proline and sugar) may involve in ROS ($H_2O_2$) homeostasis under Al stress. In proteomic analysis, over 500 protein spots were detected by 2-dimentional gel electrophoresis analysis. Total 17 Al stress-induced proteins were identified, of which 8 protein spots were up-regulated and 9 were down-regulated. The differential expression patterns of protein spots were selected and analyzed by the peptide mass fingerprinting (PMF) using MALDI-TOF MS analysis. Three protein spots corresponding to Rubisco were significantly down-regulated whereas peroxiredoxin and glutamine synthetase were up-regulated in response to Al stress. The different regulation patterns of identified proteins were involved in energy metabolism and antioxidant / ROS detoxification during Al stress in alfalfa. Taken together, these results provide new insight to understand the molecular mechanisms of alfalfa plant in terms of Al stress tolerance.

• BMB Reports
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• 제31권1호
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• pp.53-57
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• 1998

The CMCax gene from Acetobacter xylinum ATCC 23769 was cloned and expressed in E. coli. With this gene, three gene products - mature CMCax, CMCax containing signal peptide(pre-CMCax), and a glutathione-S-transferase(GST)-CMCax fusion enzyme - were expressed. CMCax and pre-CMCax are aggregated to multimeric forms which showed high CMC hydrolysis activity, whereas GST-CMCax was less aggregated and showed lower activity, indicating that oligomerization of CMCax controbutes to the cellulose hydrolysis activity to achieve greater efficiency. The enzyme was identified to be an $\beta$-1,4-endoglucanase, which catalyzes the cleavage of internal $\beta$-1,4-glycosidic bonds of cellulose. The reaction products, cellobiose and cellotriose, from cellopentaose as a substrate, were identified by HPLC. Substrate specificity of cellotetraose by this enzyme was poor, and the reaction products consisted of glucose, cellobiose, and cellotriose in a very low yield. Theses results suggested that cellopentaose might be the oligosaccharide substrate consisting of the lowest number of glucose. The optimum pH of CMCax and pre CMCax was about 4.5, whereas that of GST-CMCas was rather broad at pH 4.5-8. The physiological significance of cellulose-hydrolyzing enzyme, CMCax, having such low $\beta$-1,4-endoglucanase activity and low optimum pH in cellulose-producing A. xylinum is not clearly known yet, but it seems to be closely related to the production of cellulose.

• Animal cells and systems
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• 제11권2호
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• 한국식품조리과학회지
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• 제24권3호
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• pp.349-357
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• 2008

In an effort to augment extractability of carnosine and anserine at the levels of pro-oxidants such as iron and protein in Tuna boiled extracts(Skipjack, Yellowfin and Bigeye), we assessed the effects of heated and ion exchange chromatography(IEC) and ultrafiltration(UF) using a MW 500 cut-off(500 MWCO). We also evaluated the antioxidant activity of these extracts processed as free radical scavengers and reducing agents. Tuna boiled extracts of dark and ordinary muscle protein and total iron were reduced, whereas carnosine and anserine concentrations and antioxidant activity were increased. The carnosine and anserine concentrations of the ion exchange and permeate UF(IEC-UF) extracts were higher than those observed in the heated and permeate UF(heat-UF), whereas the protein and total iron contents were lower than that observed in the heat-UF. The quantity of carnosine and anserine in ordinary muscle was higher than that detected in dark muscle. HPLC analysis and SDS-PAGE were shown to removes the effect of UF on high molecular weight impurities in the tuna boiled extracts. The major free amino acids(FFAs) from Skipjack, Yellowfin and Bigeye tuna IEC-UF extracts were anserine, histidine and carnosine. These three peptides constituted more than 80～85%. of the detected amino acid. The IEC-UF treated ordinary muscle extracts evidenced the highest levels of DPPH radical scavenging activity and the highest levels of reducing power among the various extracts. The IEC-UF extracts evidenced a DPPH radical scavenging effect equal to that of 1mM ascorbic acid.

• 한국식품과학회지
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• 제39권6호
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• pp.625-629
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• 2007

불가사리 골편의 콜라겐으로부터 활성 펩타이드를 분리하기 위하여 초음파를 처리하여 조직을 단편화 시키고 이후 collagnease를 처리하였다. 초음파를 처리할 경우 40kHz의 경우 38.89%의 수율을 나타내었다. 이후 펩타이드의 분자량을 측정하여 12, 20.6, 24, 43, 58a, 100, 116 kDa에서 특정 밴드를 보였다. 이후 Sephadex G-75 컬럼을 이용하여 fraction 별로 모아 사용하였다. 세포 독성을 측정하고 시료 처리 후 형태학적 관찰을 동반한 결과 24 kDa의 경우 최고 농도인 1.0 mg/mL에서 26.7%를 나타내었으며 4번의 계대 이후에도 형태학적 변화가 나타나지 않아 독성이 없다고 해석할 수 있다. 이후 UVA처리 후 MMP-1의 발현을 탐색한 결과 116 kDa부터 24 kDa까지 최고농도인 1.0 mg/mL에서 40, 46.3, 56.8, 57.9, 62.4%의 control 대비 저해율을 보였다. 외부적 스트레스인 UVA에 의한 AP-1의 활성도를 증가시키는 과정을 억제한 것으로 볼 수 있으며 결과적으로 MMP-1의 발현을 효과적으로 조절한 것이라 사료되어 향후 불가사리 콜라겐 유래 펩타이드의 향장소재 활용 가능성이 높다고 할 수 있겠다.

• 농업과학연구
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• 제25권1호
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• pp.52-67
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• 1998

본 연구는 포유동물의 젖에 함유되어 있는 lactoferrin(LF)의 생리적 특성을 밝혀 기능성 식품 특히 항균작용을 이용한 식품제조업에의 이용을 위한 기초자료를 얻고자 실시하였다. 따라서 젖소의 초유로부터 lactoferrin을 분리 정제하여, 소화부위에 따른 lactoferrin의 항균활성을 분석하고자 pepsin, trypsin 및 chymotrypsin으로 분해하여 lactoferrin의 항균력을 측정하고, gel-filtration으로 분리 정제된 각각의 fraction별로 단백질을 정량하여 Escherichia coli와 Staphylococcus aureus에 접종하여 항균효과를 지니는 각 효소별 fraction의 분자량과 peptide fragment를 검토하였다. 1. 젖소의 초유로부터 분리 정제된 lactoferrin(LF)을 단백질 분해 효소인 pepsin, trypsin 및 chymotrypsin으로 분해한 결과 bovine lactoferrin은 SDS-PAGE를 수행하여 band를 확인할 수 있었다. pepsin으로 분해된 lactoferrin은 분자량 14KDa까지에서도 band를 확인할 수가 없었으며, trypsin과 chymotrypsin으로 분해한 lactoferrin은 분해되지 않은 lactoferrin이 존재함을 나타내고, 33KDa에서도 band를 확인할 수 있었다. 2. Sephadex G-50 column을 사용하여 bovine lactoferrin를 효과적으로 정제하였다. Sephadex G-50 column chromatography를 수행 한 결과 bovine lactoferrin은 Tris-HCl사이에서 용출되었으며, pepsin, trypsin 및 chymotrypsin으로 분해한 lactoferrin은 각각 2, 3, 2 개의 peak를 보였고, HPLC 분석결과 첫번째 peak는 주로 분해되지 않은 lactoferrin 수용체가 존재하는 것으로 확인되었으며, trypsin과 chymotrypsin처리에서 항균효과도 유사점을 관찰할 수 있었다. 3. 효소처리한 lactoferrin의 항균효과를 알아보기 위하여 Escherichia coli와 Staphylococcus aureus를 접종하여 항균활성을 비교하였다. 그 결과 각 효소에 대하여 pepsin으로 처리한 lactoferrin을 접종한 시험구가 대조구에 비하여 낮은 생장율을 보여 현저한 항균활성을 지님을 알 수 있었다. 그리고 trypsin과 chymotrysin에 의한 분해물도 미생물 배양 8시간까지는 효소 처리전 bovine lactoferrin보다는 항균활성을 나타냄을 알 수 있었다. 4. Sephadex G-50 column을 사용하여 분리 정제된 bovine lactoferrin fraction별로 SDS-PAGE를 실시한 결과 lactoferrin fraction의 경우는 chromatography 수행 결과와 비교하여, pepsin과 chymotrypsin 분해물은 저분자량임을 알 수 있었고, trypsin에 의한 lactoferrin만이 단일 band를 보임으로서 단백질 분해의 특징을 볼 수 있었다.

• Journal of Rheumatic Diseases
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• 제24권4호
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• pp.192-202
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• 2017

Objective. Rebampide is a gastroprotective agent used to treat gastritis. It possesses anti-inflammatory and anti-arthritis effects, but the mechanisms of these effects are not well understood. The objective of this study was to explore mechanisms underlying the therapeutic effects of rebamipide in inflammatory arthritis. Methods. Collagen-induced arthritis (CIA) was induced in DBA/1J mice. DBA/1J mice were immunized with chicken type II collagen, then treated intraperitoneally with rebamipide (10 mg/kg or 30 mg/kg) or vehicle (10% carboxymethylcellulose solution) alone. Seven weeks later, plasma samples were collected. Plasma metabolic profiles were analyzed using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolomics study and metabolite biomarkers were identified through multivariate data analysis. Results. Low dose rebamipide treatment reduced the clinical arthritis score compared with vehicle treatment, whereas high dose rebamipide in CIA aggravated arthritis severity. Based on multivariate analysis, 17 metabolites were identified. The plasma levels of metabolites associated with fatty acids and phospholipid metabolism were significantly lower with rebamipide treatment than with vehicle. The levels of $15-deoxy-^{{\Delta}12,14}$ prostaglandin J2 and thromboxane B3 decreased only in high dose-treated groups. Certain peptide molecules, including enterostatin (VPDPR) enterostatin and bradykinin dramatically increased in rebamipide-treated groups at both doses. Additionally, corticosterone increased in the low dose-treated group and decreased in the high dose-treated group. Conclusion. Metabolomics analysis revealed the anti-inflammatory effects of rebamipide and suggested the potential of the drug repositioning in metabolism- and lipid-associated diseases.

• 한국초지조사료학회지
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• 제31권2호
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• pp.99-106
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• 2011

프로테오믹스 기법을 이용하여 벼 고온 스트레스 관련 단백질을 분리 동정하기 위하여 $42^{\circ}C$에서 고온처리한 벼의 줄기로부터 단백질을 분리하였다. 분리한 단백질로부터 Rubisco 단백질을 제거하기 위해 15% PEG fractionation을 실시한 후 상등액 분획의 단백질을 이차원전기 영동한 후, CBB 염색을 통해 차별적 발현을 보이는 단백질을 분석하였다. 총 46개의 단백질 spot이 발현양에 변화를 보였으며, 그 중 24개의 단백질이 고온 스트레스에 의해 발현이 증가되었으며, 22개의 단백질이 감소하는 발현 양상을 나타내었다. 이들 단백질을 MALDI-TOF MS와 database를 통해 동정한 결과 에너지 대사관련 단백질, 산화 환원 관련 단백질 및 저분자량 small HSP 등, 10개의 단백질이 동정되었다. 이들 동정된 단백질들은 식물의 고온 스트레스에 대한 적응기작을 이해하는데 중요한 단서를 제공할 것이며, 특히 미토콘드리아 small HSP는 프로테옴 분석법에 의해 최초로 동정되었으며, 금후 내하고성 목초 분자육종에 활용될 수 있는 좋은 유전자로 판단된다.

• 한국잡초학회지
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• 제12권4호
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• pp.368-373
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• 1992

귀리에 alachlor를 처리(處理)할 때 변화(變化)되는 단백질(蛋白質)을 분석(分析)한 결과요약(結果要約)하면 다음과 같다. Alachlor 처리시(處理時) 단백질(蛋白質) 합성(合成)은 $1{\times}10^{-4}$M에서 5.8%, $1{\times}10^{-3}$M에서는 86.5% 억제(抑制)되는 현상(現象)을 보였지만 $1{\times}10^{-5}$M 이하(以下)의 농도(濃度)에서는 증가(增加)되는 현상(現象)을 보였다. 귀리의 근단분열조직(根端分裂組織)에서 추출(抽出)된 단백질(蛋白質)은 100 kd 이하(以下)의 polypeptide로 구성(構成)되어 있으며, alachlor 처리시(處理時) 47kd 이상(以上)의 고분자(高分子) 단백질(蛋白質)들은 억제(抑制)되었지만 23kd 이하(以下)의 저분자(低分子) 단백질(蛋白質)들은 오히려 증가(增加)되는 현상(現象)을 보였다. 이차원적(二次元的) 전기영동(電氣泳動) 결과(結果) alachlor 처리시(處理時) 83kd의 1, 2 spot, 70kd의 3, 4 spot, 47.5kd의 5, 6 spot의 polypeptide가 억제(抑制)된 반면(反面), 20kd의 7 spot, 16kd 의 8, 9, 10 spot는 증가(增加)되는 현상(現象)을 보였다. 또한 귀리 근단(根端) 분열조직(分裂組織)은 산성(酸性) 단백질(蛋白質)로서, 주(主)로 중성(中性) 부위(部位)에 큰 polypeptide spots들이 존재(存在)하고 있으며, 작은 spots을은 산성(酸性)쪽에 분리(分離)되어 나타났다.