• Title/Summary/Keyword: Response Characterization

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Biochemical Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG), Using CHO Cells and PathHunter Parental Cells Expressing Equine Luteinizing Hormone/Chorionic Gonadotropin Receptors (eLH/CGR) (말의 LH/CGR를 발현하는 CHO 세포와 PathHunter Parental 세포에서 유전자 재조합 eCGβ/α의 생화학적 특성)

  • Lee, So-Yun;Byambaragchaa, Munkhzaya;Kim, Jeong-Soo;Seong, Hun-Ki;Kang, Myung-Hwa;Min, Kwan-Sik
    • Journal of Life Science
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    • v.27 no.8
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    • pp.864-872
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    • 2017
  • Equine chorionic gonadotropin (eCG) consists of highly glycosylated ${\alpha}-$ and ${\beta}-subunits$ and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of $rec-eCG{\beta}/{\alpha}$, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of $rec-eCG{\beta}/{\alpha}$ in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. $rec-eCG{\beta}/{\alpha}$ was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the $rec-eCG{\beta}/{\alpha}$ protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of $rec-eCG{\beta}/{\alpha}$. The cAMP concentration increased in direct proportion to the concentration of the $rec-eCG{\beta}/{\alpha}$. The $EC_{50}$ value in the transient transfected CHO-K1 cells was $8.1{\pm}6.5ng$. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing ${\beta}-arrestin$. We found that $rec-eCG{\beta}/{\alpha}$ had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The $EC_{50}$ value in transient and stable cells was $5.0{\pm}4.7ng/ml$ and $4.5{\pm}5.2ng/ml$, respectively. These results suggest that $rec-eCG{\beta}/{\alpha}$ has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated $rec-eCG{\beta}/{\alpha}$ mutants.

Application of LCA Methodology on Lettuce Cropping Systems in Protected Cultivation (시설재배 상추에 대한 전과정평가 (LCA) 방법론 적용)

  • Ryu, Jong-Hee;Kim, Kye-Hoon
    • Korean Journal of Soil Science and Fertilizer
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    • v.43 no.5
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    • pp.705-715
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    • 2010
  • The adoption of carbon foot print system is being activated mostly in the developed countries as one of the long-term response towards tightened up regulations and standards on carbon emission in the agricultural sector. The Korean Ministry of Environment excluded the primary agricultural products from the carbon foot print system due to lack of LCI (life cycle inventory) database in agriculture. Therefore, the research on and establishment of LCI database in the agriculture for adoption of carbon foot print system is urgent. Development of LCA (life cycle assessment) methodology for application of LCA to agricultural environment in Korea is also very important. Application of LCA methodology to agricultural environment in Korea is an early stage. Therefore, this study was carried out to find out the effect of lettuce cultivation on agricultural environment by establishing LCA methodology. Data collection of agricultural input and output for establishing LCI was carried out by collecting statistical data and documents on income from agro and livestock products prepared by RDA. LCA methodology for agriculture was reviewed by investigating LCA methodology and LCA applications of foreign countries. Results based on 1 kg of lettuce production showed that inputs including N, P, organic fertilizers, compound fertilizers and crop protectants were the main sources of major emission factor during lettuce cropping process. The amount of inputs considering the amount of active ingredients was required to estimate the actual quantity of the inputs used. Major emissions due to agricultural activities were $N_2O$ (emission to air) and ${NO_3}^-$/${PO_4}^-$ (emission to water) from fertilizers, organic compounds from pesticides and air pollutants from fossil fuel combustion in using agricultural machines. The softwares for LCIA (life cycle impact assessment) and LCA used in Korea are 'PASS' and 'TOTAL' which have been developed by the Ministry of Knowledge Economy and the Ministry of Environment. However, the models used for the softwares are the ones developed in foreign countries. In the future, development of models and optimization of factors for characterization, normalization and weighting suitable to Korean agricultural environment need to be done for more precise LCA analysis in the agricultural area.

Effect of Soil Temperature on the Emergence - Speed of Rice and Barnyardgrasses under Dry Direct - Seeding Condition (토양온도(土壞溫度)가 벼와 피의 출아속도(出芽速度)에 미치는 영향(影響))

  • Kwon, Y.W.;Kim, D.S.;Park, S.W.
    • Korean Journal of Weed Science
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    • v.16 no.2
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    • pp.81-87
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    • 1996
  • Seeds of rice, cv. Ilpoom, and barnyardgrasses(Echinochloa crus-galli, vars. oryzicola, crux-galli, and praticola) were sown for a characterization of their responses to temperature during emergence under a dry direct-seeded condition. A laboratory-made aluminum block apparatus for emergence-temperature control conferred a linear continuous temperature gradient from 10 to $30^{\circ}C$ to the seeds from cooling to heating ends of the apparatus. The lowest temperature for emergence was $12.3^{\circ}C$ for rice cv. Ilpoom, and $11.0^{\circ}C$ for the three varieties of Echinochloa spp.. Percent emergence of rice increased sharply with an increase in temperature by ca. $20^{\circ}C$, then leveled-off, while those of barnyardgrasses increased almost linearly with temperatures up to $30^{\circ}C$. In rice the time required for emergence after seeding was shortened exponentially with increased temperature while those for barnyardgrasses were shortened almost linearly from 11 to $30^{\circ}C$. The temperature-response characteristic of rice in emergence-speed was almost the same among those for the 1st emergence, emergence by 25, 50, 75%, or average emergence time. At $13^{\circ}C$, $346.7^{\circ}C$ days of accummulated temperature(26.67 days) were required for the 1st emergence in rice while 131.7, 136.0, and $138.7^{\circ}C$ days(10.13, 10.46, and 10.67 days) were required for the 1st emergence in E. spp., vars. crus-galli, praticola, and oryzicola, respectively. Greater cold tolerance and increasingly faster emergence of barnyardgrasses than rice below $20^{\circ}C$ seem to render the barnyardgrasses as much more competitive than rice at lower temperatures.

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Isolation and characterization of sigH from Corynebacterium glutamicum (Corynebacterium glutamicum의 sigH 유전자의 분리 및 기능분석)

  • Kim Tae-Hyun;Kim Hyung-Joon;Park Joon-Sung;Kim Younhee;Lee Heung-Shick
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.99-104
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    • 2005
  • Corynebacterial clones which exert regulatory effects on the expression of the glyoxylate bypass genes were isolated using a reporter plasmid carrying the enteric lacZ fused to the aceB promoter of Corynebacterium glutamicum. Some clones carried common fragments as turned out by DNA mapping technique. Subcloning analysis followed by the measurement of $\beta-galactosidase$ activity in Escherichia coli identified the region responsible for the aceB-repressing activity. Sequence analysis of the DNA fragment identified two independent ORFs of ORF1 and ORF2. Among them, ORF2 was turned out to be responsible for the aceB-repressing activity. ORF1 encoded a 23,216 Da protein composed of 206 amino acids. Sequence similarity search indicated that the ORF may encode a ECF-type $\sigma$ factor and designated sigH. To identify the function of sigH, C. glutamicum sigH mutant was constructed by gene disruption technique and the sigH mutant showed growth retardation as compared to the wild type strain. In addition, the mutant strain showed sensitivity to oxidative-stress generating agent plumbagin. This result imply that sigH is probably involved in the stress response occurring during normal cell growth.

Isolation and Characterization of a Novel Transcription Factor ATFC Activated by ER Stress from Bombyx mori Bm5 Cell Lines (누에 배양세포(Bm5)로부터 분리한 새로운 전사제어인자 ATFC의 특성분석)

  • 구태원;윤은영;김성완;최광호;황재삼;박수정;권오유;강석우
    • Journal of Life Science
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    • v.13 no.5
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    • pp.596-603
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    • 2003
  • Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding molecular chaperones and folding enzymes. The information is transmitted from the ER lumen to the nucleus by intracellular signaling pathway, called the unfolded protein response (UPR). To obtain genes related to UPR from B. mori, the cDNA library was constructed with mRNA isolated from Bm5 cell lines in which N-glycosylation was inhibited by tunicamycin treatment. From the cDNA library, we selected 40 clones that differentially expressed when cells were treated with tunicamycin. Among these clones, we have isolated ATFC gene showing similarity with Hac1p, encoding a bZIP transcription factor of 5. cerevisiae. Basic-leucine zipper (bZIP) domain in amino acid sequences of ATFC shared homology with yeast Hac1p. Also, ATFC is up-regulated by accumulation of unfolded proteins in the ER through the treatment of ER stress drugs. Therefore we suggest that ATFC represents a major component of the putative transcription factor responsible for the UPR leading to the induction of ER-localized stress proteins.

Molecular Characterization of Cinnamate 4-Hydroxylase gene in Red Hot Pepper (Capsicum annuum L.) (고추에서 분리한 Cinnamate 4-Hydroxylase 유전자의 분자생물학적 특성)

  • Kim Kye-Won;Ha Sun-Hwa;Cho Kang-Jin;Kim Eun-Ju;Lee Min-Kyung;Yu Jae-Ju;Kim Jong-Guk;Lee Shin-Woo
    • Journal of Plant Biotechnology
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    • v.32 no.3
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    • pp.167-173
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    • 2005
  • Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

Characterization of bft Genes among Enterotoxigenic Bacteroides fragilis Isolates from Extraintestinal Specimens at a University Hospital in Korea (국내 한 대학병원의 임상검체에서 분리된 Bacteroides fragilis 독소 유전자의 특성)

  • Kim, Myungsook;Kim, Hyunsoo;Ji, Seung Eun;Rim, John Hoon;Gwon, Sun Yeong;Kim, Wan Hee;Rhee, Ki-Jong;Lee, Kyungwon
    • Korean Journal of Clinical Laboratory Science
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    • v.48 no.2
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    • pp.82-87
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    • 2016
  • Enterotoxigenic Bacteroides fragilis (ETBF) produces enterotoxins known to be a virulence factor. Three isotypes of the B. fragilis toxin (BFT) gene have been identified: bft-1, bft-2, and bft-3. We investigated the presence of bft isotypes in clinical B. fragilis isolates and the antimicrobial resistance of BFT-negative and BFT-positive isolates. Overall, 537 B. fragilis isolates were collected from extraintestinal specimens over 8 years (2006~2013) from a university hospital in Korea. Samples were analyzed by multiplex PCR to identify the bft gene isotypes. Additionally, the antimicrobial susceptibility of 107 B. fragilis isolates (74 BFT-negative and 33 BFT-positive) was examined by the CLSI agar dilution method. PCR revealed a total bft gene detection rate of 30%, while 33% and 29% of blood and other extraintestinal isolates contained the gene, respectively. Among ETBF isolates, the most common isotype was bft-1 gene, followed by bft-2 and bft-3 (bft-1 77%, bft-2 14%, bft-3 9%). Resistance rates (%) for BFT-negative and positive isolates differed in response to various antimicrobial agents, with 3%, 5%, 1% and 38% of BFT-negative isolates and 3%, 6%, 3% an 42% of BFT-positive isolates being resistant to piperacillin-tazobactam, cefoxitin, imipenem, and clindamycin, respectively. Interestingly, neither BFT-negative nor positive isolates showed antimicrobial resistance to chloramphenicol and metronidazole. Overall, the proportion of ETBF from blood was similar to that of other extraintestinal sites and the bft-1 gene was the predominant isotype. Higher antimicrobial resistance rates were found in BFT-positive isolates than BFT-negative isolates, but these differences were not statistically significant.

Quantitative Microbial Risk Assessment of Pathogenic Vibrio through Sea Squirt Consumption in Korea (우렁쉥이에 대한 병원성 비브리오균 정량적 미생물 위해평가)

  • Ha, Jimyeong;Lee, Jeeyeon;Oh, Hyemin;Shin, Il-Shik;Kim, Young-Mog;Park, Kwon-Sam;Yoon, Yohan
    • Journal of Food Hygiene and Safety
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    • v.35 no.1
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    • pp.51-59
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    • 2020
  • This study evalutated the risk of foodborne illness from Vibrio spp. (Vibrio vulnificus and Vibrio cholerae) through sea squirt consumption. The prevalence of V. vulnificus and V. cholerae in sea squirt was evaluated, and the predictive models to describe the kinetic behavior of the Vibrio in sea squirt were developed. Distribution temperatures and times were collected, and they were fitted to probabilistic distributions to determine the appropriate distributions. The raw data from the Korea National Health and Nutrition Examination Survey 2016 were used to estimate the consumption rates and amount of sea squirt. In the hazard characterization, the Beta-Poisson model for V. vulnificus and V. cholerae infection was used. With the collected data, a simulation model was prepared and it was run with @RISK to estimate probabilities of foodborne illness by pathogenic Vibrio spp. through sea squirt consumption. Among 101 sea squirt samples, there were no V. vulnificus positive samples, but V. cholerae was detected in one sample. The developed predictive models described the fates of Vibrio spp. in sea squirt during distribution and storage, appropriately shown as 0.815-0.907 of R2 and 0.28 of RMSE. The consumption rate of sea squirt was 0.26%, and the daily consumption amount was 68.84 g per person. The Beta-Poisson model [P=1-(1+Dose/β)] was selected as a dose-response model. With these data, a simulation model was developed, and the risks of V. vulnificus and V. cholerae foodborne illness from sea squirt consumption were 2.66×10-15, and 1.02×10-12, respectively. These results suggest that the risk of pathogenic Vibrio spp. in sea squirt could be considered low in Korea.

Analysis of Characterization in Commercial Extra Virgin Olive Oils (유통 압착올리브유의 이화학적인 특성)

  • Nam, Ha-Young;Lee, Ki-Teak
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.36 no.7
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    • pp.866-873
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    • 2007
  • To analyze and differentiate volatile compounds of 13 extra virgin olive oils from market, solid-phase micro extraction (SPME) GC-MS and electronic nose (EN) equipped with metal oxide sensors were applied. The volatiles identified in extra virgin olive oils include hexanal, 4-hexen-1-ol, (Z)-3-hexen-1-ol, acetic acid, and 2,4-dimethyl-heptane, etc. Response from EN was analysed by the principal component analysis. Proportion of the first Principal component was 99.70%, suggesting that each aroma pattern of the 13 extra virgin olive oils could be discriminated by EN. Fatty acid compositions were oleic (61.1${\sim}$77.9 mole%), palmitic (11.7${\sim}$16.5 mole%), linoleic (4.7${\sim}$9.7 mole%), stearic (2.5${\sim}$2.9 mole%), Palmitoleic (0.8${\sim}$2.4 mole%), and linolenic acid (0.7${\sim}$1.2 mole%). In color study, extra virgin olive oil showed $L^{\ast}$ value of 81.7${\sim}$92.9, $a^{\ast}$ value of -28.3${\sim}$13.5 and $b^{\ast}$ value of 52.2${\sim}$139.0. Total phenol and ${\alpha}-tocopherol$ contents were 6.2${\sim}$24.9 mg/100 g and 5.5${\sim}$12.8 mg/100 g, respectively. In Rancimat test, the induction period of 13 extra virgin olive oils showed 31.76${\sim}$54.04 hr while their POV ranged from 13.5 to 22.9 meq/kg oil.

Molecular Characterization and Expression Analysis of Clathrin-Associated Adaptor Protein 3-δ Subunit 2 (AP3S2) in Chicken

  • Oh, Jae-Don;Bigirwa, Godfrey;Lee, Seokhyun;Song, Ki-Duk
    • Korean Journal of Poultry Science
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    • v.46 no.1
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    • pp.31-37
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    • 2019
  • A chicken clathrin-associated adaptor protein $3-{\delta}$ subunit 2 (AP3S2) is a subunit of AP3, which is involved in cargo protein trafficking to target membrane with clathrin-coated vesicles. AP3S2 may play a role in virus entry into host cells through clathrin-dependent endocytosis. AP3S2 is also known to participate in metabolic disease developments of progressions, such as liver fibrosis with hepatitis C virus infection and type 2 diabetes mellitus. Chicken AP3S2 (chAP3S2) gene was originally identified as one of the differentially expressed genes (DEGs) in chicken kidney which was fed with different calcium doses. This study aims to characterize the molecular characteristics, gene expression patterns, and transcriptional regulation of chAP3S2 in response to the stimulation of Toll-like receptor 3 (TLR3) to understand the involvement of chAP3S2 in metabolic disease in chicken. As a result, the structure prediction of chAP3S2 gene revealed that the gene is highly conserved among AP3S2 orthologs from other species. Evolutionarily, it was suggested that chAP3S2 is relatively closely related to zebrafish, and fairly far from mammal AP3S2. The transcriptional profile revealed that chAP3S2 gene was highly expressed in chicken lung and spleen tissues, and under the stimulation of poly (I:C), the chAP3S2 expression was down-regulated in DF-1 cells (P<0.05). However, the presence of the transcriptional inhibitors, BAY 11-7085 (Bay) as an inhibitor for nuclear factor ${\kappa}B$ ($NF{\kappa}B$) or Tanshinone IIA (Tan-II) as an inhibitor for activated protein 1 (AP-1), did not affect the expressional level of chAP3S2, suggesting that these transcription factors might be dispensable for TLR3 mediated repression. These results suggest that chAP3S2 gene may play a significant role against viral infection and be involved in TLR3 signaling pathway. Further study about the transcriptional regulation of chAP3S2 in TLR3 pathways and the mechanism of chAP3S2 upon virus entry shall be needed.