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IKKγ Facilitates the Activation of NF-κB by Hsp90 (Hsp90에 의한 NF-κB의 활성화를 촉진하는 IKKγ의 역할)

  • Lee, Jeong Ah;Kim, Dong Wan
    • Journal of Life Science
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    • v.32 no.3
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    • pp.241-248
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    • 2022
  • NF-κB acts as a critical transcription factor in inflammation and innate immunity, and it is also closely involved in cell survival and tumorigenesis via induction of anti-apoptotic genes. In these processes, NF-κB cooperates with multiple other signaling molecules and pathways, and although many studies have demonstrated that Hsp90 regulates NF-κB activity, the exact mechanism is unclear. In this study, we investigated the relationship between Hsp90 and IKKγ in the regulation of NF-κB using expression plasmids of IKK complex components. Wild-type and deletion mutants of IKKγ were expressed together with Hsp90, and the combined regulatory effect of Hsp90 and IKKγ on NF-κB activation was assayed. The results show that Hsp90 activates NF-κB by promoting the phosphorylation and degradation of IκBα and that activation of NF-κB by NIK and LPS was increased by Hsp90. IKKγ elevated the effect of Hsp90 on NF-κB activation by increasing phosphorylation and degradation of IκBα. The positive regulation on NF-κB by Hsp90 and IKKγ was also proved in analysis with IKKβ-EE, the constitutively active form of IKKβ. In experiments with the deletion mutants of IKKγ, the N-terminal IKKβ binding domain, C-terminal leucine zipper, and zinc finger domains of IKKγ were found not necessary for the positive regulation of NF-κB activity. Additionally, the expression of pro-inflammatory cytokines was synergistically elevated by Hsp90 and IKKγ. These results indicate that inhibiting the interaction between Hsp90 and IKKγ is a possible strategic method for controlling NF-κB and related diseases.

20(S)- Protopanaxadiol suppresses hepatic stellate cell activation via WIF1 demethylation-mediated inactivation of the Wnt/β-catenin pathway

  • Chunxue Li ;Yating Zhan ;Rongrong Zhang;Qiqi Tao ;Zhichao Lang ;Jianjian Zheng
    • Journal of Ginseng Research
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    • v.47 no.4
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    • pp.515-523
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    • 2023
  • Background: 20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. Methods: The anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. Results: PPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl4)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. Conclusion: PPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the downregulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

Identification of relevant differential genes to the divergent development of pectoral muscle in ducks by transcriptomic analysis

  • Fan Li;Zongliang He;Yinglin Lu;Jing Zhou;Heng Cao;Xingyu Zhang;Hongjie Ji;Kunpeng Lv;Debing Yu;Minli Yu
    • Animal Bioscience
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    • v.37 no.8
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    • pp.1345-1354
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    • 2024
  • Objective: The objective of this study was to identify candidate genes that play important roles in skeletal muscle development in ducks. Methods: In this study, we investigated the transcriptional sequencing of embryonic pectoral muscles from two specialized lines: Liancheng white ducks (female) and Cherry valley ducks (male) hybrid Line A (LCA) and Line C (LCC) ducks. In addition, prediction of target genes for the differentially expressed mRNAs was conducted and the enriched gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes signaling pathways were further analyzed. Finally, a protein-to-protein interaction network was analyzed by using the target genes to gain insights into their potential functional association. Results: A total of 1,428 differentially expressed genes (DEGs) with 762 being up-regulated genes and 666 being down-regulated genes in pectoral muscle of LCA and LCC ducks identified by RNA-seq (p<0.05). Meanwhile, 23 GO terms in the down-regulated genes and 75 GO terms in up-regulated genes were significantly enriched (p<0.05). Furthermore, the top 5 most enriched pathways were ECM-receptor interaction, fatty acid degradation, pyruvate degradation, PPAR signaling pathway, and glycolysis/gluconeogenesis. Finally, the candidate genes including integrin b3 (Itgb3), pyruvate kinase M1/2 (Pkm), insulin-like growth factor 1 (Igf1), glucose-6-phosphate isomerase (Gpi), GABA type A receptor-associated protein-like 1 (Gabarapl1), and thyroid hormone receptor beta (Thrb) showed the most expression difference, and then were selected to verification by quantitative real-time polymerase chain reaction (qRT-PCR). The result of qRT-PCR was consistent with that of transcriptome sequencing. Conclusion: This study provided information of molecular mechanisms underlying the developmental differences in skeletal muscles between specialized duck lines.

The Variation of Natural Population of Pinus densiflora S. et Z. in Korea (III) -Genetic Variation of the Progeny Originated from Mt. Chu-wang, An-Myon Island and Mt. O-Dae Populations- (소나무 천연집단(天然集團)의 변이(變異)에 관(關)한 연구(硏究)(III) -주왕산(周王山), 안면도(安眠島), 오대산(五臺山) 소나무집단(集團)의 차대(次代)의 유전변이(遺傳變異)-)

  • Yim, Kyong Bin;Kwon, Ki Won
    • Journal of Korean Society of Forest Science
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    • v.32 no.1
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    • pp.36-63
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    • 1976
  • The purpose of this study is to elucidate the genetic variation of the natural forest of Pinus densiflora. Three natural populations of the species, which are considered to be superior quality phenotypically, were selected. The locations and conditions of the populations are shown in table 1 and 2. The morphological traits of tree and needle and some other characteristics were presented already in our first report of this series in which population and family differences according to observed characteristics were statistically analyzed. Twenty trees were sampled from each populations, i.e., 60 trees in total. During the autumn of 1974, matured cones were collected from each tree and open-pollinated seeds were extracted in laboratory. Immediately after cone collection, in closed condition, the morphological characteristics were measured. Seed and seed-wing dimensions were also studied. In the spring of 1975, the seeds were sown in the experimental tree nursery located in Suweon. And in the April of 1976, the 1-0 seedlings were transplanted according to the predetermined experimental design, randomized block design with three replications. Because of cone setting condition. the number of family from which progenies were raised by populations were not equal. The numbers of family were 20 in population 1. 18 in population 2 and 15 in population 3. Then, each randomized block contained seedlings of 53 families from 3 populations. The present paper is mainly concerned with the variation of some characteristics of cone, seed, needle, growth performance of seedlings, and chlorophyll and monoterpene compositions of needles. The results obtained are summerized as follows. 1. The meteorological data obtained by averaging the records of 30 year period, observed from the nearest station to each location of populations, are shown in Fig. 3, 4, and 5. The distributional pattern of monthly precipitation are quite similar among locations. However, the precipitation density on population 2, Seosan area, during growing season is lower as compared to the other two populations. Population 1. Cheong-song area, and population 3, Pyong-chang area, are located in inland, but population 2 in the western seacoast. The differences on the average monthly air temperatures and the average monthly lowest temperatures among populations can hardly be found. 2. Available information on the each mother trees (families) studied, such as age, stem height, diameter at breast height, clear-bole-length, crown conditions and others are shown in table 6,7, and 8. 3. The measurements of fresh cone weight, length and the widest diameter of cone are given in Tab]e 9. All these traits arc concerned with the highly significant population differences and family differences within population. And the population difference was also found in the cone-index, that is, length-diameter ratio. 4. Seed-wing length and seed-wing width showed the population differences, and the family differences were also found in both characteristics. Not discussed in this paper, however, seed-wing colours and their shapes indicate the specificity which is inherent to individual trees as shown in photo 3 on page 50. The colour and shape are fully the expression of genetic make up of mother tree. The little variations on these traits are resulted from this reason. The significant differences among populations and among families were found in those characteristics, such as 1000-seed weight, seed length, seed width, and seed thickness as shown in table 11. As to all these dimensions, the values arc always larger in population 1 which is younger in age than that of the other two. The population differences evaluated by cone, seed and seed-wing sizes could partly be attributed to the growth vigorousity. 5. The values of correlation between the characteristics of cone and seed are presented in table 12. As shown, the positive correlations between cone diameter and seed-wing width were calculated in all populations studied. The correlation between seed-wing length and seed length was significantly positive in population 1 and 3 but not in population 2, that is, the r-value is so small as 0.002. in the latter. The correlation between cone length and seed-wing length was highly significant in population 1, but not in population 2. 6. Differences among progenies in growth performances, such as 1-0 and 1-1 seedling height and root collar diameter were highly singificant among populations as well as families within population(Table 13.) 7. The heritability values in narrow sense of population characteristics were estimated on the basis of variance components. The values based on seedling height at each age stage of 1-1 and 1-0 ranged from 0.146 to 0.288 and the values of root collar diameter from 0.060 to 0.130. (Table 14). These heritability values varied according to characteristics and seedling ages. Here what must be stated is that, for calculation of heritability values, the variance values of population was divided by the variance value of environment (error) and family and population. The present authors want to add the heritability values based on family level in the coming report. It might be considered that if the tree age is increased in furture, the heritability value is supposed to be altered or lowered. Examining the heritability values studied previously by many authors, in pine group at age of 7 to 15, the values of height growth ranged from 0.2 to 0.4 in general. The values we obtained are further below than these. 8. The correlation between seedling growth and seed characteristics were examined and the values resulted are shown in table 16. Contrary to our hypothetical premise of positive correlation between 1-0 seedling height and seed weight, non-significance on it was found. However, 1-0 seedling height correlated positively with seed length. And significant correlations between 1-0 and 1-1 seedling height are calculated. 9. The numbers of stomata row calculated separately by abaxial and adaxial side showed highly significant differences among populations, but not in serration density. On serration density, the differences among families within population were highly significant. (Table 17) A fact must be noted is that the correlation between stomata row on abaxial side and adaxial side was highly significant in all populations. Non-significances of correlation coefficient between progenies and parents regarding to stomata row on abaxial side were shown in all populations studied.(Table 18). 10. The contents of chhlorophyll b of the needle were a little more than that of chlorophyll a irrespective of the populations examined. The differences of chlorophyll a, b and a plus b contents were highly significant but not among families within populations as shown in table 20. The contents of chlorophyll a and b are presented by individual trees of each populations in table 21. 11. The occurrence of monoterpene components was examined by gas liquid chromatography (Shimazu, GC-1C type) to evaluate the population difference. There are some papers reporting the chemical geography of pines basing upon monoterpene composition. The number of populations studied here is not enough to state this problem. The kinds of monoterpene observed in needle were ${\alpha}$-pinene, camphene, ${\beta}$-pinene, myrcene, limonene, ${\beta}$-phellandrene and terpinolene plus two unknowns. In analysis of monoterpene composition, the number of sample trees varied with population, I.e., 18 families for population 1, 15 for population 2 and 11 for population3. (Table 22, 23 and 24). The histograms(Fig. 6) of 7 components of monoterpene by population show noticeably higher percentages of ${\alpha}$-pinene irrespective of population and ${\beta}$-phellandrene in the next order. The minor Pinus densiflora monoterpene composition of camphene, myrcene, limonene and terpinolene made up less than 10 percent of the portion in general. The average coefficients of variation of ${\alpha}$-pinene and ${\beta}$-phellandrene were 11 percent. On the contrary to this, the average coefficients of variation of camphene, limonene and terpinolene varied from 20 to 30 percent. And the significant differences between populaiton were observed only in myrcene and ${\beta}$-phellandrene. (Table 25).

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Effect of Air Temperature on Growth and Phytochemical Content of Beet and Ssamchoo (온도처리가 비트와 쌈추의 생육과 생리활성 물질 함량에 미치는 영향)

  • Lee, Sang Gyu;Choi, Chang Sun;Lee, Hee Ju;Jang, Yoon Ah;Lee, Jun Gu
    • Horticultural Science & Technology
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    • v.33 no.3
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    • pp.303-308
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    • 2015
  • The consumption of leaf vegetables has been steadily increasing in Korea. Leaf vegetables are used for "Ssam (vegetable wrap-up), eaf vegetables has been steadily increasing in Korea. Leaf vegetables are used for asoned condiments inside several layers of young vegetable leaves. This study investigated the effect of air temperature on the growth and phytochemical contents of beet (Beta vulgaris L.) and Ssamchoo (Brassica lee L. ssp. namai) grown in a closed-type plant factory system where fluorescent lamps were used as an artificial light source. Seeds of beet and Ssamchoo were sown in a peat-lite germination mix. The roots of 20-day-old seedlings were washed, and the seedlings were planted on a styrofoam board and grown in hydroponic beds for 25 days under fluorescent light. Plants were exposed to one of three different air temperature regimes (20, 25 and $30^{\circ}C$ during the day combined with $18^{\circ}C$ during the night), which were monitored with a sensor at 30 cm above the plant canopy. Increased plant height and leaf area were observed in beet at $25^{\circ}C$ and $30^{\circ}C$ compared to $20^{\circ}C$. For Ssamchoo, the greatest plant height, leaf area, fresh weight and dry weight were obtained at $20^{\circ}C$. Ascorbic acid content of beet and Ssamchoo leaves were highest at $30^{\circ}C$. In beet, total polyphenol and flavonoid contents were higher at $20^{\circ}C$ (42.4, $197.0mg{\cdot}g^{-1}DW$) and $25^{\circ}C$ (46.9, $217.0mg{\cdot}g^{-1}DW$) than $30^{\circ}C$ (22.4, $88.0mg{\cdot}g^{-1}DW$). In Ssamchoo, total polyphenol and flavonoid contents were also higher at $20^{\circ}C$ (79.2, $268.2mg{\cdot}g^{-1}DW$) and $25^{\circ}C$ (66.3, $258.3mg{\cdot}g^{-1}DW$), respectively, than $30^{\circ}C$ (53.7, $134.7mg{\cdot}g^{-1}DW$). Hence, the optimum temperature appears to be $20^{\circ}C$ for growing both beet and Ssamchoo in a closed-type plant factory system with fluorescent light.

A Study on Relationships Between Environment, Organizational Structure, and Organizational Effectiveness of Public Health Centers in Korea (보건소의 환경, 조직구조와 조직유효성과의 관계)

  • Yun, Soon-Nyoung
    • Research in Community and Public Health Nursing
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    • v.6 no.1
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    • pp.5-33
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    • 1995
  • The objective of the study are two-fold: one is to explore the relationship between environment, organizational structure, and organizational effectiveness of public health centers in Korea, and the other is to examine the validity of contingency theory for improving the organizational structure of public health care agencies, with special emphasis on public health nursing administration. Accordingly, the conceptual model of the study consisted of three different concepts: environment, organizational structure, and organizational effectiveness, which were built up from the contingency theory. Data were collected during the period from 1st of May through 30th of June, 1990. From the total of 249 health centers in the country, one hundred and five centers were sampled non proportionally, according to the geopolitical distribution. Out of 105, 73 health centers responded to mailed questionnaire. The health centers were the unit of the study, and a various statistical analysis techniques were used: Reliability analysis(Cronbach's Alpha) for 4 measurement tools; Shapiro-Wilk statistic for normality test of measured scores of 6 variables: ANOVA, Pearson Correlaion analysis, regressional analysis, and canonical correlation analysis for the test of the relationships and differences between the variables. The results were. as follows : 1. No significant differences between forma lization, decision-making authority and environmental complexity were found(F=1.383, P=.24 ; F=.801, P=.37). 2. Negative relationships between formalization and decision-making authority for both urban and rural health centers were found(r=-.470, P=.002 ; r=-.348, P=.46). 3. No significant relationship between formalization and job satisfaction for both urban and rural health centers were found (r=-.242, P=.132, r=-.060, P=.739). 4. Significant positive relationship between decision - making authority and job satisfaction were found in urban health centers (r=.504, P=.0009), but no such relationship was observed in rural health centers. Regression coefficient between them was statistically significant($\beta=1.535$, P=.0002), and accuracy of regression line was accepted (W=.975, P= .420). 5. No significant relationships among formalization and family planning services, maternal health services, and tuberculosis control services for both urban and rural health centers were found. 6. Among decision-making authority and family planning services, maternal health services, and tuberculosis control services, significant positive relationship was found between de cision-making authority and family planning services(r=.286, P=.73). 7. A significant difference was found in maternal health services by the type of health centers (F=5.13, P=.026) but no difference was found in tuberculosis control services by the type of health centers, formalization, and decision-making authority. 8. A significant positive relationships were found between family planning services and maternal health services and tuberculosis control services, and between maternal health services and tuberculosis control services (r=-.499, P=.001 ; r=.457, P=.004 ; r=.495, P=.002) in case of urban health centers. In case of rural health centers, relationships between family planning services and tuberculosis control services, and between maternal health services and tuberculosis control services were statistically significant (r=.534, P=.002 ; r=.389, P=.027). No significant relationship was found between family planning and maternal health services. 9. A significant positive canonical correlation was found between the group of independent variables consisted of formalization and de cision-making authority and the group of dependent variables consisted of family planning services, maternal health services and tuberculosis control services(Rc=.455, P=.02). In case of urban health centers, no significant canonical correlation was found between them, but significant canoncial correlation was found in rural health centers(Rc=.578, P=.069), 10. Relationships between job satisfaction and health care productivity was not found significant. Through these results, the assumed relationship between environment and organizational structure was not supported in health centers. Therefore, the relationship between the organizational effectiveness and the congruence between environment and organizational structure that contingency theory proposes to exist was not able to be tested. However decision-making authority was found as an important variable of organizational structure affecting family planning services and job satisfaction in urban health centers. Thus it was suggested that decentralized decision making among health professionals would be a valuable strategy for improvement of organizational effectiveness in public health centers. It is also recommended that further studies to test contingency theory would use variability and uncertainty to define environment of public health centers instead of complexity.

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The Differences of the Smoking Habit Between Emphysema and Chronic Bronchitis (폐기종과 만성기관지염의 흡연습관 차이)

  • Mun, Yeung-Chul;Yu, Sung-Keun;Park, Hye-Jung;Shin, Kyeong-Cheol;Chung, Jin-Hong;Lee, Kwan-Ho;Lee, Jung-Cheol
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.6
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    • pp.693-703
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    • 2001
  • Background : Smoking is the most important and consistent determinant of the development and progression of COPD(Ed Note : Define COPD). The fact that cigarette smokers develop a different type of COPD, chronic bronchitis and emphysema, with different clinical and pathological aspects, suggests that the development of COPD has a relationship with other smoking-associated factors beyond just a simple smoking history. The aim of this was to analyze the smoking habits and history of patients with COPD and to evaluate the development of different types of COPD according to patient's smoking habits. Method : To evaluate the differences in the smoking patterns of patients with chronic bronchitis and emphysema, a pulmonary function test was conducted, and the smoking history and patterns was obtained through a smoking history questionnaire by a direct personal interview from 333 male cigarette smokers diagnosed with COPD, in the Yeungnam university medical center(190 patients diagnosed with chronic bronchitis, 143 patients diagnosed with emphysema). Result : The patients with emphysema smoked earlier and had a higher smoking history(ie, more packyears, more total amounts of smoked cigarette, and more deep inhalation and longer duration of plain cigarette exposure) than those with chronic bronchitis. The depth of inhalation was also significantly higher in the emphysema patients after taking into account age, cumulative cigarette consumption and the type of cigarette smoked. Conclusion : Emphysema was more associated with the increasing degree of inhalation as assessed by the depth of inhalation. A high alveolar smoke exposure may be a significant risk factor for the development of emphysema.

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Development of System-Wide Functional Analysis Platform for Pathogenicity Genes in Magnaporthe oryzae

  • Park, Sook-Young;Choi, Jaehyuk;Choi, Jaeyoung;Kim, Seongbeom;Jeon, Jongbum;Kwon, Seomun;Lee, Dayoung;Huh, Aram;Shin, Miho;Jung, Kyungyoung;Jeon, Junhyun;Kang, Chang Hyun;Kang, Seogchan;Lee, Yong-Hwan
    • 한국균학회소식:학술대회논문집
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    • 2014.10a
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    • pp.9-9
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    • 2014
  • Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.

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Total Polyphenol Contents and Antioxidant Activities of Methanol Extracts from Vegetables produced in Ullung Island (울릉도산 산채류 추출물의 총 폴리페놀 함량 및 항산화 활성)

  • Lee, Syng-Ook;Lee, Hyo-Jung;Yu, Mi-Hee;Im, Hyo-Gwon;Lee, In-Seon
    • Korean Journal of Food Science and Technology
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    • v.37 no.2
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    • pp.233-240
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    • 2005
  • To discover new functional materials using edible plants, antioxidant activities of methanol extracts from various parts of seven wild vegetables were investigated in vitro. Total polyphenol contents, determined by Folin-Denis method, varied from 16.74 to $130.22{\mu}g/mg$. Radical-scavenging activities of methanol extracts were examined using ${\alpha},\;{\alpha}-diphenyl-{\beta}-pirrylhydrazyl$ (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay. Inhibition effects on peroxidation of linoleic acid determined by ferric thiocyanate (FTC) method and on oxidative degradation of 2-deoxy-D-ribose in Fenton-type reaction system were dose-dependent. Athyrium acutipinulum Kodama (leaf and rood), Achyranthes japonica (Miq.) Nakai (seed), and Solidago virga-aurea var. gigantea Nakai (root) showed relatively high antioxidant activities in various systems.

A Study on the Morphological Structure of Sasul-Sijo (사설시조의 형태구조 연구)

  • Won, Yong-Moon
    • Sijohaknonchong
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    • v.23
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    • pp.161-188
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    • 2005
  • The purpose of this study was to delve into the morphological types of Sijo in an effort to determine the morphological structure of Sasul-sijo, and it's also attempted to present standard about how to discriminate Pyong-si, Eos-sijo and Sasul-sijo from one another from a morphological standpoint. It's suggested that Si with tee Jangs, six verses and 12 stanzas or more, with three Jangs, seven verses and 14 stanzas or more, and with three Jangs, eight verses and 16 stanzas or more should respectively be called Pyong-sijo, Eos-sijo and Sasul-sijo. After what Sijo was and what's not were discussed, how to distinguish Eos-sijo from Sasul-sijo was described, and finally, the structure of Sasul-sijo was presented. As for Sijo and non-Sijo, the types of works that consisted of tee Jangs, like Sijo, yet didn't suit its framework and Yuljo and were written in Chinese characters were regarded as non-Sijo. Concerning discrimination between Eos-si and Sasul-sijo, the type of Sijo that included one more or higher number of verse(s) and two more or higher number of stanzas in one of three Jangs was defined as Eos-sijo, and the type of Sijo that involved two more or higher number of verses and four more or higher number of stanzas in one of three Jangs was called Sasul-sijo. In other words, Eos-sijo contained one more verse in one of tee Jangs, and Sasul-sijo included one more Jang in one tee Jangs. The sort of Sijo that contained one more Jang in one of three Jangs could be viewed as Sasul-sijo. Regarding the structure of Sasul-si, there should be three Jangs, eight verses and 16 stanzas in one piece of Sasul-sijo. Any type of Sijo that contained two more or higher number of verses and four more or higher number of stanzas could be called Sasul-sijo. Such an addition of verse and stanza could done in various ways. The examples were (1) adding stanzas the first Jang, 2) adding stanzas to the second Jang, (3) adding stanzas to the final Jang, (4) adding stanzas to both the first and Second Jangs, (5) adding stanzas to th the second and final Jangs, and (6) adding stanzas to all the first, second and third Jangs at the same time. Besides, there was an extremely broad gap between the numbers of verse and stanza in Sasul-sijo, which ranged from a low of eight stanzas to a high of 87 ones in one of three Jangs.

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