• Title/Summary/Keyword: Recombinant

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Expression of Transforming Growth Factor-$\alpha$ and Transforming Growth Factor-$\beta$ In Human Primary Lung Cancers (인체 폐암종의 TGF-$\alpha$ 및 TGF-$\beta$의 발현에 관한 면역 조직화학적 연구)

  • Lew, Woo-Jin;Shin, Dong-Ho;Park, Sung-Soo;Lee, Dong-Hoo;Lee, Jung-Dal;Lee, Jung-Hee
    • Tuberculosis and Respiratory Diseases
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    • v.42 no.4
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    • pp.492-501
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    • 1995
  • Background: Transforming growth factor- alpha(TGF-$\alpha$) may play important roles in carcinogenesis, tumor growth, and angiogenesis. Transforming growth factor-beta(TGF-$\beta$) are known to be involved in cell-cycle control and regeneration. TGF-$\alpha$ positively acts on growth control of many epithelial cells in contrast to the negative role of TGF-$\beta$. Method: To evaluate the possible role of TGF-$\alpha$ and TGF-$\beta$ in human primary lung cancers, the expression of TGF-$\alpha$ and TGF-$\beta$ were immmunohistochemically investigated in tissue sections from forty seven cases with lung cancers and ten cases with non-cancerous lung tissues. Recombinant cloned monoclonal antibody of TGF-$\alpha$ and neutralizing antibody of TGF-$\beta$ were employed as primary antibodies after dewaxing the formalin-fixed, paraffinized tissue sections. Results: TGF-$\alpha$ was expressed in the cytoplasms of tumor cells in thirty five cases of forty seven(74.5%) primary lung cancers, whereas the control expressed in two of ten brochial epithelial cells. The expression of TGF-$\alpha$ was disclosed in four cases of eleven(36.4 %) small cell carcinomas and thirty one cases of thirty six(86.1%) non-small cell carcinomas of the lung. Expressions of TGF-$\beta$ was discernible in bronchial epithelium in eight of ten non-cancerous lung tissues. The expression of TGF-$\beta$ was noted in the cytoplasms of tumor cells in eight cases of forty seven(17.0%) primary lung cancers. The expression of TGF-$\beta$ disclosed in two cases of eleven(18.2%) small cell carcinomas and six cases of thirty six(16.7%) non- small cell carcinomas of the lung. Conclusion: These findings suggest that up-regulation of TGF-$\alpha$ and down-regulation of TGF-$\beta$ are involved during development and growth of primary lung cancers.

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Diagnostic Significance of the Serologic Test Using Antigen of Mycobacterium Tuberculosis for Antibody Detection by ELISA (결핵항원에 대한 혈청학적 검사와 진단적 유용성)

  • Park, Jae-Min;Park, Yeon-Soo;Chang, Yeon-Soo;Kim, Young-Sam;Ahn, Kang-Hyun;Kim, Se-Kyu;Chang, Joon;Kim, Sung-Kyu;Lee, Won-Young;Cho, Shang-Rae
    • Tuberculosis and Respiratory Diseases
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    • v.45 no.2
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    • pp.271-279
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    • 1998
  • Background: Diagnosis by direct microscopy and/or by culture of the Mycobacterium tuberculosis from body fluids or biopsy specimens is "Gold standard". However, the sensitivity of direct microscopy after Ziehl-Neelsen staining is relatively low and culture of mycobacteria is time consuming. Detection of mycobacterial DNA in clinical samples by the polymerase chain reaction is highly sensitive but laborious and expensive. Therefore, rapid, sensitive and readily applicable new tests need to be developed. So we had evaluated the clinical significance of serologic detection of antibody to 38 kDa antigen, which is known as the most specific to the M. tuberculosis complex, and culture filtrate antigen by ELISA in sputum AFB smear negative patients. Method: In this study, culture tests for acid fast bacilli with sputa or bronchial washing fluids of 183 consecutive patients who were negative of sputum AFB smear were performed. Simultaneously serum antibodies to 38 kDa antigen and unheated culture filtrate of M. tuberculosis were detected by an ELISA method. Results: The optical densities of ELISA test with 38 kDa and culture filtrate antigen were significantly higher in active pulmonary tuberculosis cases than in non tuberculous pulmonary diseases (p<0.05), but in patients with active pulmonary tuberculosis, those of the sputum culture positive patients for M. tuberculosis were not significantly different from those of the sputum culture negative cases(p>0.05). In the smear-negative active pulmonary tuberculosis patients, the sensitivity of the ELISA using 38 kDa antigen and culture filtrate was 20.0% and 31.4%. respectively. The specificity was 95.3% and 93.9%. respectively. Conclusion : In active pulmonary tuberculosis but smear negative, the serologic detection of antibody to 38 kDa antigen and culture filtrate by ELISA cannot substitute traditional diagnostic tests and does not have clinically significant role to differenciate the patient with active pulmonary tuberculosis from other with non-tuberculous pulmonary diseases.

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Radioimmunoassay for Determination of Serum Macrophage Migration Inhibitory Factor (혈중 대식세포 유주 저지 인자 측정을 위한 방사면역측정법)

  • Lee, Tae-Sup;Shin, Seok-Hwan;Song, Jee-In;Woo, Kwang-Sun;Chung, Wee-Sup;Choi, Chang-Woon;Lim, Sang-Moo
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.6
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    • pp.532-539
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    • 2004
  • Purpose: There has been a renewal of interest in Macrophage migration inhibitory factor (MIF), especially correlation in pathogenesis of sepsis by many infectious diseases and in regulation of host inflammatory and immune response. We developed immunoradiometric assay (IRMA) to determine serum human MIF concentration. Materials and Methods: The IRMA system utilizes solid phase bound monoclonal anti-recombinant human MIF (rhMIF) antibody as a capture antibody, biotinylated polyclonal anti-rhMIF antibody as a detector antibody. We applied with rhMIF that concentration of standard solutions increased from 0 ng/ml to 100 ng/ml. We used $^{125}I$-streptavidin (SA) as radiotracer to determination of rhMIF concentration. Streptavidin was labeled with $^{125}I$ by Chloramine-T method and $^{125}I$-SA was purified by ultracentrifugation. $^{125}I$-SA stability was evaluated by ITLC analysis at $4^{\circ}C$ and room temperatures until 60days. To validate IRMA system for MIF, we experimented intra-assay and inter-assay coefficients of variation, recovery test and dilution test. Results: Radiolabeling yield of $^{125}I$-SA was 87% and purified $^{125}I$-SA retained above 99% radiochemical purity. $^{125}I$-SA showed above 93% stability in $4^{\circ}C$ until 60days that it is good for immunoradiometric assay as radiotracer. Plotted standard dose response curve showed that increased concentration of rhMIF linearly correlated (R2=0.99) with bound radioactivity of $^{125}I$-SA. The highest intra- and inter-assay coefficients of variation were 5.5% and 7.6%, respectively. The average of recovery of MIF in samples was 102%. In dilution test, linear response curves were obtained (R2=0.97). Conclusion: Radioimmunoassay using $^{125}I$-SA as radiotracer thought to be useful for the determination of serum MIF concentration, and further, its data will be used to evaluate the correlation between clinical significance and serum MIF concentration in patients with various inflammatory diseases.

Cytotoxicity of lymphokine activated peritoneal macrophages against Trichomonas vaginalis (질트리코모나스에 대한 림포카인황성대식세포의 세포독성능)

  • Yoon, Kyong;Ryu, Jae-Sook;Min, Duk-Young
    • Parasites, Hosts and Diseases
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    • v.29 no.4
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    • pp.381-388
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    • 1991
  • Trichomonas vaginalis is a parasitic nagellate in the urogenital tract of human. Innate cytotonicity of macrophages against T. vaginalis has been recognized, but any report on the cytotoxicity of Iymphokine-activated macrophages to T vaginalis is not yet available. The present study aimed to elucidate the Iymphokine-activated cell mediated cytotoxic effect against T. vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured by counting the release of $^3H-thymidine$ from prelabeled protozoa, and tested in U-bottom microtiter plates. Nitrite concentration in culture supernatants was measured by standard Griess reaction. The results obtained are as follows: 1, The cytotoxicity of macrophages was increased by addition of rIL-2 or $rIFN-{\gamma}$$. 2, Cytotoxicity of macrophages was reduced by addition of rIL-4 to rOM-CSV, rIL-2 or $rIFN-{\gamma}$. 3. Crude Iymphokine mixed with anti-lL-2 decreased the cytotoxity of macrophages. 4. In case of macrophages cultured with $rIFN-{\gamma}$ or rIL-4, the concentration of nitrite was related with cytotokity of macrophages against T. vaginalis, but the cytotoxicity of macrophages cultured with rIL-2 and $rIFN-{\gamma}$ was decreased in spite of its high production of llitrite. From the results obtained, it is assumed that rIL-2 and $rIFN-{\gamma}$ enhance the cytotoxicity of macrophages while rIL-4 inhibits the cytotoxicity against T. vaginalis, and that the production of nitrite does not relate with the cytotoxicity of macrophages, but nitric oxide may play a role as an inhibitory factor on the proliferation of T. vaginalis.

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Effects of Pseudomonas fluorescens on Production of Several Inflammatory Mediators in the Human Alveolar Epithelial Cells. (재조합 단백질 생산에 이용되는 Pseudomonas fluorescens의 인체 폐포 상피세포의 염증성 인자들의 발현에 미치는 영향)

  • Yang, Hyun;Ryoo, Jung-Min;Park, Seung-Hwan;Choi, Hye-Jin;Kim, Na-Yeon;Cho, Hyung-Hoon;Ahn, Jung-Hoon;Moon, Yu-Seok
    • Journal of Life Science
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    • v.18 no.4
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    • pp.530-536
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    • 2008
  • To investigate the molecular mechanism of the airway inflammation by Pseudomonas fluorescens, effects on the inflammatory mediators such as interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), macophage inhibitory cytokine 1 (MIC-1) were assessed in the human alveolar epithelial cells. Exposure to P. fluorescens and its recombinant bacteria suppressed cellular viability in the A549 epithelial cells and pro-inflammatory cytokine interleukin-8 production. However, pro-inflammatory prostaglandin-producing COX-2 protein was not altered by P. fluorescens though its mRNA was slightly elevated. As the inhibitory cytokine for the pro-inflammatory mediators, MIC-1 expression was monitored in A549 cells. MIC-1 gene induction was not significantly enhanced but the protein processing was changed by exposure to P. fluorescens. Pro-protein form of MIC-1 (${\sim}40\;kD$) was cleaved into active form mature MIC-1 (${\sim}15\;kD$) and propeptide (${\sim}28\;kD$) by the bacteria exposure. MIC-1 activation can contribute to the suppression of cellular viability by P. fluorescens and can retard IL-8-induced monocyte recruitment. However, sustained activation of MIC-1 can mediate the tissue injury by P. fluorescens exposure.

Disease Reaction of a Japonica Rice, Keumo3, and Detection of a Linked DNA Marker to Leaf Blast Resistance ("금오3호"의 벼 잎도열병 저항성 특성 및 저항성 연관 마커 탐색)

  • Lee, Jong-Hee;Kwak, Do-Yeon;Pakr, Dong-Soo;Roh, Jae-Hwan;Kang, Jong-Rae;Kim, Choon-Song;Jeon, Myeong-Gi;Yeo, Un-Sang;Yi, Gihwan;Shin, Mun-Sik;Oh, Byeong-Geun;Hwang, Hung-Goo
    • Korean Journal of Breeding Science
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    • v.40 no.4
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    • pp.408-413
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    • 2008
  • Rice blast resistance is considered one of the most important traits in rice breeding and the disease, caused by Magnaporthe grisea Barr, has brought significant crop losses annually. Moreover, breakdown of resistance normally occurs in two to five years after cultivar release, thus a more durable resistance is needed for better control of this disease. We developed a new variety, Keumo3, which showed strong resistance to leaf blast. It was tested in 2003 to 2007 at fourteen blast nursery sites covering entire rice-growing regions of South Korea. It showed resistance reactions in 12 regions and moderate in 2 regions without showing susceptible reactions. Durability test by sequential planting method indicated that this variety had better resistance. Results showed that Keumo3 was incompatible against 19 blast isolates with the exception of KI101 by artificial inoculation. To understand the genetic control of blast resistance in rice cultivar Keumo3 and facilitate its utilization, recombinant inbred lines (RIL) consisting of 290 F5 lines derived from Akidagomachi/Keumo3 were analyzed and genotyped with Pizt InDel marker zt56591. The recombination value between the marker allele of zt56591 and bioassay data of blast nursery test was 1.1%. These results indicated that MAS can be applied in selecting breeding populations for blast resistance using zt56591 as DNA marker.

Identification of Leaf Blast Resistance Genes Derived from a Korean Weedy Rice, Ganghwaaengmi 11 (잡초성벼인 강화앵미11 유래 잎도열병 저항성 유전자 탐색)

  • Suh, Jung-Pil;Cho, Young-Chan;Kim, Jeong-Ju;Shin, Young-Seop;Yang, Chang-Ihn;Roh, Jae-Hwan;Kim, Yeon-Gyu
    • Korean Journal of Breeding Science
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    • v.42 no.4
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    • pp.390-396
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    • 2010
  • A weedy rice, Ganghwaaengmi 11, shows high level of leaf blast resistance. The chromosomal number and locations of genes conferring the leaf blast resistance were detected by QTL (quantitative trait loci) analysis using SSR markers in the 120 RILs (recombinant inbred lines) derived from the cross between Nagdongbyeo and Ganghwaaengmi 11. Ganghwaaengmi 11 expressed compatibility with 20 of the 45 inoculated blast isolates, in contrast to Nagdongbyeo with 44 compatible isolates. To identify QTLs affecting partial resistance, RILs were assessed in upland blast nursery in three regions and inoculated with selected nine blast isolates. QTLs for resistance to blast isolates were identified on chromosomes 7, 11 and 12. Three QTLs associated with blast resistance in nursery test at three regions were also detected on chromosomes 7, 11 and 12. The QTL commonly detected on chromosome 12 was only increased blast resistance by Ganghwaaengmi 11 allele. This QTL accounted for 60.3~78.6% of the phenotypic variation in the blast nursery test. OSR32 and RM101 markers tightly linked to QTL for blast resistance on chromosome 12 might be useful for marker-assisted selection (MAS) and gene pyramiding to improve the blast resistance of japonica rice.

Analysis of Transcriptional Activity and Estrogen Responsiveness of Regulatory Elements in Chicken Ovalbumin Promoter (닭 오브알부민 프로모터의 길이에 따른 유전자 발현 활성 및 에스트로겐 반응성 분석)

  • Yang, Hyeon;Kim, Kyung-Woon;Kim, Jeom Sun;Woo, Jae-Seok;Lee, Hwi-Cheul;Choi, Hoonsung;Jung, Sun Keun;Sureshkumar, Shanmugam;Lee, Haesun;Oh, Keon Bong;Byun, Sung June
    • Korean Journal of Poultry Science
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    • v.46 no.1
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    • pp.17-24
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    • 2019
  • Chickens have been considered as well-defined animal bioreactor. The optimized ovalbumin promoter is essential for recombinant protein production in transgenic chicken. Here we try to compare the activity and identify the effect of estrogen on ovalbumin promoter according to each promoter length with estrogen response element (ERE) existence. We cloned two (2.8 and 5.5 kb) ovalbumin promoters that the 5.5 kb contained the ERE but the 2.8 kb did not, and these two promoters were cloned to pGL4.11 vector. Additionally, we constructed another pGL4.11 vector containing of the 4.4 kb (with ERE) ovalbumin promoter deleted with 1 kb between ERE region and the 2.8 kb promoter. For reporter assay, HeLa, MES-SA, LMH/2A, and cEF cells were transfected with all the pGL4.11 vectors. The comparative analysis showed that the mutated 4.4 kb promoter has more potent activity than the 2.8 and 5.5 kb promoters in HeLa, MES-SA, and LMH/2A cells. However, there is no significant difference in cEFs. Also, these cells transfected with the mutated 4.4 kb promoter were treated with the $17{\beta}$-estradiol (0~3,000 nM) and HeLa, MES-SA, and LMH/2A cells showed estrogen responsibilities, but cEFs did not. Besides, the mutated 4.4 kb promoter has still higher activity than the 2.8 and 5.5 kb promoter, and there is no transcriptional induction effect in 2.8 kb promoter at 500 nM estrogen that is blood concentration of laying hens. Hence our study strongly suggested that the mutated 4.4 kb promoter is considered as one of the most efficient length for generating transgenic chicken.

Identification of DNA Markers Related to Resistance to Herbicide Containing Mesotrione in Tongil Type Rice (통일형 벼에서 메소트리온계 제초제 저항성 연관 DNA marker 탐색)

  • Lee, Ji-Yoon;Cho, Jun-Hyeon;Lee, Jong-Hee;Cho, Su-Min;Kwon, Young-Ho;Park, Dong-Soo;Song, You-Chun;Ko, Jong-Min
    • Korean Journal of Breeding Science
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    • v.50 no.4
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    • pp.387-395
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    • 2018
  • This study was conducted to identify DNA markers related to resistance to herbicide containing mesotrione in Tongil type rice. Two Tongil type elite lines; Milyang154 and Suweon382, showed resistance to mesotrione, whereas the others were susceptible at 20 days after mesotrione application, and severe growth inhibition was observed in the remaining 13 lines. As a result of analysis of mesotrione resistance using 190 $F_2$ populations derived from a cross of Hanareum2 (susceptible) and Milyang154 (resistant), the mesotrione resistance locus was shown to be a single dominant gene with a 3:1 segregation ratio ($X^2=1.19$, P=0.31). To identify a DNA marker closely linked to the mesotrione resistance gene, bulked segregant analysis (BSA) was adopted. The DNA marker RM3501 was identified on chromosome 2 with a recombinant value of 0.53 to the mesotrione resistance gene. Mst1(t) was located between SSR (simple sequence repeat) markers RM3501 and RM324 with a physical map distance of 10.2 Mb-11.4 Mb on chromosome 2. The band pattern of agarose gel electrophoresis of the SSR marker RM3501 showed the same segregation pattern with respect to mesotrione treatment in 20 Tongil type varieties and a $BC_2F_2$ segregation population derived from a cross between Unkwang (resistant) and Hanareum2 (susceptible). Thus, the RM3501 DNA marker could be used in breeding programs for Marker Assisted Selection in mesotrione resistant rice breeding.

Confirmation of Enzymatic Synthesis of 1, 2-Octanediol Galactoside using Mass Spectrometry and NMR Spectroscopy (Mass spectrometry와 NMR Spectroscopy를 이용한 1, 2-Octanediol Galactoside의 효소합성 확인)

  • Lee, Hyang-Yeol;Jin, Hong-Jong;An, Seung Hye;Lee, Hye Won;Jung, Kyung-Hwan
    • Journal of the Korean Applied Science and Technology
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    • v.38 no.3
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    • pp.824-831
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    • 2021
  • 1, 2-Octanediol galactoside (OD-gal) has been synthesized from 1, 2-octanediol (OD), as a safer cosmetic preservative, using recombinant Escherichia coli β-galactosidase (β-gal). To confirm the molecular structure of synthesized OD-gal, mass spectrometry and NMR (1H- and 13C-) spectroscopy of OD-gal were carried out. In the reaction mixture, a sodium adduct ion of OD-gal (m/z=331.1732) was identified using mass spectrometry analysis. In addition, 1H NMR spectrum of OD-gal showed multiple peaks corresponding to the galactosyl group, which is evidence of galactosylation on OD. Downfield proton peaks at δH 4.39 ppm and multiple peaks from δH 3.98~3.55 ppm were indicative of galactosylation on OD. Up field proton peaks at δH 1.52~1.26 ppm and 0.89 ppm showed the presence of CH2 and CH3 protons of OD. 13C NMR spectrum revealed the presence of 24 carbons suggestive of α- and β-anomers of OD-gal. Among 14 carbon peaks from each anomer, the 4 peaks at δC 31.4, 29.0, 22.3, and 13.7 ppm were assigned to be overlapped showing only 24 peaks out of a total of 28 peaks. The mass value from mass spectrometry analysis of OD-gal, and 1H and 13C NMR spectral data were in good agreement with the expecting structure of OD-gal. Finally, we identified a galactose molecule from the hydrolysate of OD-gal using β-gal. We are expecting that through future study it will eventually be able to develop a safe cosmetic preservative.