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Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach

  • Shin, Jong-Hwan;Han, Joon-Hee;Park, Hyun-Hoo;Fu, Teng;Kim, Kyoung Su
    • The Plant Pathology Journal
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    • 제35권6호
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    • pp.575-584
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    • 2019
  • Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH4Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 ㎍/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

Characterization of a QTL associated with chlorophyll content using progeny from an interspecific cross in rice (Oryza Sativa L.)

  • Shim, Kyu-Chan;Luong, Ngoc Ha;Kim, Sun Ha;Jeon, Yun-A;Lu, Xin;Ahn, Sang-Nag
    • 한국작물학회:학술대회논문집
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    • 한국작물학회 2017년도 9th Asian Crop Science Association conference
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    • pp.23-23
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    • 2017
  • Rice (Oryza sativa L.) is the world's most important cereal crop. In crop plant, chlorophyll content and leaf senescence could affect grain filling and yield. We analyzed a QTL associated with chlorophyll content and delayed leaf senescence using high chlorophyll near isogenic line (HC-NIL). HC-NIL derived from a cross between Oryza sativa cv. Hwaseong as a recurrent parent and wild species O. grandiglumis as a donor parent showed higher chlorophyll content than Hwaseong. To identify QTL associated with chlorophyll content, 58 $F_3$ and 38 $F_4$ lines were developed from a cross between HC-NIL and Hwaseong. For QTL analysis, simple sequence repeat (SSR) markers were used for genotyping and one-way ANOVA was conducted. A QTL for chlorophyll content (qCC2) was detected in chromosome 2 and explained 24.63% of phenotypic variation. The senescence effect of the qCC2 was examined in dark-induced incubation (DII). Detached leaves from Hwaseong and HC-NIL were incubated on 3mM MES buffer (pH 5.8) at $27^{\circ}C$ under complete dark condition. After 3 days of incubation, the Hwaseong leaves turned yellow, but the HC-NIL leaves were green. HC-NIL has higher chlorophyll content with delayed senescence than Hwaseong. These results indicated that qCC2 is associated with stay-green phenotype. To know whether the qCC2 is responsible for leaf functionality, ion leakage test and Fv/Fm measurement were performed. Both experiment results showed that differences were observed between Hwaseong and HC-NIL but it was not statistically significant. These results might suggest that the qCC2 is possibly related to chlorophyll content and non-functional stay-green phenotype.

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D-Pinitol의 유방암 증식 및 재발 억제 효능 (Inhibitory Effect of D-pinitol on Both Growth and Recurrence of Breast Tumor from MDA-MB-231 Cancer Cells)

  • 김윤섭;박지성;김민지;황방연;이종길;송석길
    • 생약학회지
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    • 제45권2호
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    • pp.174-180
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    • 2014
  • D-Pinitol, an anti-diabetic substance, is a naturally occurring compound found in legumes. In this study, we investigated the inhibitory effect of D-pinitol on growth and recurrence of breast cancer. When D-pinitol was treated on MDA-MB-231 or MCF-7 breast cancer cells, it was observed that the viability of the two cancer cell lines was reduced in MTT assay. In order to examine the effect on the growth of breast tumor, mouse xenograft assay was carried out. On day 0, nine millions cells of MDA-MB-231 were injected subcutaneously into nude mouse and D-pinitol was administered orally at the dose of 500 mg/kg or 1000 mg/kg body weight for consecutive 45 days. Tumor size was reduced in dose-dependent manner upto 95.4% in 1000 mpk-treated group, compared with the non-treated control group. When D-pinitol was co-administrated with $4{\mu}g$ of doxorubicin, recurrence of breast tumor was delayed by two weeks, compared with the mouse group of doxorubicin monotherapy. Consistent with this data, it was observed that the population of cancer stem cells (CSCs), responsible for recurrence of cancer, within tumor mass was significantly reduced. Taken together, D-pinitol inhibits the growth of breast cancer and relapse of the tumor by suppressing the proliferation of CSCs.

Determination of mortar strength using stone dust as a partially replaced material for cement and sand

  • Muhit, Imrose B.;Raihan, Muhammad T.;Nuruzzaman, Md.
    • Advances in concrete construction
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    • 제2권4호
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    • pp.249-259
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    • 2014
  • Mortar is a masonry product which is matrix of concrete. It consists of binder and fine aggregate and moreover, it is an essential associate in any reinforced structural construction. The strength of mortar is a special concern to the engineer because mortar is responsible to give protection in the outer part of the structure as well as at a brick joint in masonry wall system. The purpose of this research is to investigate the compressive strength and tensile strength of mortar, which are important mechanical properties, by replacing the cement and sand by stone dust. Moreover, to minimize the increasing demand of cement and sand, checking of appropriateness of stone dust as a construction material is necessary to ensure both solid waste minimization and recovery by exchanging stone dust with cement and sand. Stone dust passing by No. 200 sieve, is used as cement replacing material and retained by No. 100 sieve is used for sand replacement. Sand was replaced by stone dust of 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50% by weight of sand while cement was replaced by stone dust of 3%, 5%, and 7% by weight of cement. Test result indicates that, compressive strength of specimen mix with 35% of sand replacing stone dust and 3% of cement replacing stone dust increases 21.33% and 22.76% respectively than the normal mortar specimen at 7 and 28 days while for tensile it increases up to 13.47%. At the end, optimum dose was selected and crack analysis as well as discussion also included.

Effect of chloroform extract of traditional Dicranopteris linearis leaves against paracetamol- and CCl4-induced liver toxicity in rats

  • Din, Syafawati Shamsahal;Mamat, Siti Syariah;Ismail, Noor Aisyah;Zainulddin, Wan Noraziemah Wan;Zabidi, Zalina;Yahya, Farhana;Kamisan, Farah Hidayah;Mohtarrudin, Norhafizah;Othman, Fezah;Suhaili, Zarizal;Zakaria, Zainul Amiruddin
    • 셀메드
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    • 제2권4호
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    • pp.34.1-34.5
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    • 2012
  • The present study aimed to determine the hepatoprotective activity of the chloroform extract of D. linearis leaves (CEDL) using the paracetamol (PCM)- and carbon tetrachloride ($CCl_4$)-induced liver injury models in rats. The rats received $dH_2O$ (negative control), 200 mg/kg of silymarin (positive control) or CEDL (50, 250 and 500 mg/kg) orally once daily for 7 days and then were subjected to the hepatotoxic induction on the $7^{th}$ day. The samples (i.e. blood and liver) were collected and underwent biochemical and microscopical analysis, respectively. From the data obtained, both inducers caused significant (p < 0.05) increase in the levels of AST and ALT when compared to the control group, which were significantly (p < 0.05) reduced by CEDL in a generally dose-dependent manner. These biochemical findings were supported by the histopathological analysis and histological scoring. In conclusion, CEDL possesses potential hepatoprotective activity, which could be associated with its flavonoid and tannin contents with the mechanisms of hepatoprotection linked to either its antioxidant or anti-inflammtory/immunomodulating activities. Further in-depth studies are required to identify the responsible bioactive compound.

Effects of Vitamin A on the Antioxidant Systems of the Growing Chicken

  • Surai, P.F.;Kuklenko, T.V.
    • Asian-Australasian Journal of Animal Sciences
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    • 제13권9호
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    • pp.1290-1295
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    • 2000
  • The present study was conducted to evaluate effects of the increased dietary vitamin A supplementation on the vitamin A, vitamin E and ascorbic acid concentrations in the plasma and liver and activities of some enzymes in the liver of the growing chicken. One hundred and twenty female chickens at 4 weeks of age were divided in 6 equal groups in accordance with their body weight. They were housed in cages and fed on standard wheat-barley-based broiler diet balanced in the major nutrients. Vitamin A was supplemented in the form of retinyl acetate. Control diet was supplemented with 10 IU/g and experimental feeds were supplemented with 50, 100, 500, 1000 and 2000 IU/g. At days 42 and 56 of the development 8 chickens from each group were killed, plasma and liver were collected for vitamin and enzyme analyses. The increased vitamin A supplementation was associated with its increased accumulation in the liver and with a reduction of ${\alpha}-tocopherol$ concentrations in the plasma and liver. The blood plasma was more resistant to vitamin A concentration changes and the retinol level was elevated only when the vitamin A dose exceeded 100 IU/g feed. Ascorbic acid concentration in the liver was elevated when moderately high vitamin A supplementation was used but significantly decreased at the highest vitamin A dose. Similar changes were observed with glycogen concentration in the liver. Activities of hexokinase, glucose-6-phosphatase and lactate dehydrogenase in the chicken liver were also dependent on vitamin A supplementation, decreasing with highest vitamin A doses. Therefore the observations showed that the vitamin A excess compromises antioxidant system of the growing chickens suggesting that prooxidant activity may be responsible for at least part of the toxicity of vitamin A.

Association of polymorphisms in Pit-1 gene with growth and feed efficiency in meat-type chickens

  • Jin, Sihua;He, Tingting;Yang, Lei;Tong, Yucui;Chen, Xingyong;Geng, Zhaoyu
    • Asian-Australasian Journal of Animal Sciences
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    • 제31권11호
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    • pp.1685-1690
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    • 2018
  • Objective: The pituitary specific transcription factor-1 (Pit-1) gene is responsible for pituitary development and growth hormone expression and is regarded as a pivotal candidate gene for growth and production in chickens. Therefore, the aim of this study was to investigate the association of Pit-1 polymorphisms with growth and feed efficiency traits in yellow meat-type chickens. Methods: In the present study, five single nucleotide polymorphisms (SNPs) of Pit-1 were selected and genotyped by high-throughput matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in 724 meat-type chickens. Results: Association analysis showed that rs13687126 of Pit-1 was strongly associated with body weight gain (BWG) and feed intake (FI) (p<0.05), and that rs13687128 was significantly correlated with body weight at 70 days of age (BW70), BWG and feed conversion ratio (FCR) (p<0.05). SNP rs13905622 was strongly related to BW70 and FCR (p<0.05). Furthermore, birds with the GG genotype of rs13687126 had larger BWG and FI than those with the AG genotype (p<0.05). Individuals with the TT genotype of rs13687128 were significantly higher BW70 and BWG than those of the CT and CC genotype, while FCR was just the opposite (p<0.05). For rs13905622, the AA chickens showed strongly larger BW70 and lower FCR compared with the AT and TT chickens (p<0.05). Additionally, an ACA haplotype based on rs13687126, rs13687128, and rs13905622 had significant effects on BW70 and FCR (p<0.05). Conclusion: Our studies thus provide crucial evidence for the relationship between polymorphisms of Pit-1 and growth and feed efficiency traits which may be useful for meat-type chicken breeding programs.

n-3 지방산이 유방암세포의 증시과 지질과산화 및 Oncogene 발현에 미치는 영향 (Effects of n-3 Fatty Acids on Proliferation of Human Breast Cancer Cells in Relatino to Lipid Peroxidation and Oncogene Expression)

  • 조성희
    • Journal of Nutrition and Health
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    • 제30권8호
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    • pp.987-994
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    • 1997
  • To investigate the effects of n-3 fatty acids on breast cancer, MDA-MB231 human breast cancer cells were cultured in the presence of $\alpha$-linolenic (LNA), eicosapentaenoic(EPA), and docosahexaenoic acid (DHA) at a concentration of 0.5$\mu\textrm{g}$/ml in serum -free IMM medium. Cell growth was monitored and thiobarbituric acid reactive substances (TBARS), $\alpha$-tocopherol contents, and oncogene expression were measured. To compare the effects of n-3 fatty acids with other types of fatty acid, steraic (STA), olieic(OA). linoleic acid(LA) were used. After one day , cell growth was retarded most highly when DHA was in the medium. Cellular TBARS level measured after three days of culture was the highest with DHA in the medium and was also increased by LNA and EPA, compared with STA, OA and LA. Alpha-tocoopherol contents of cells were decreased by DHA but only modestly. There was non significant difference in $\alpha$-tocopherol contents in cells cultured in the presence of the other fatty acids. northern blot hybridization carried out with cells cultured during 24 hours showed that levels of erbB-2 mRNA were not altered by six different fatty acids in the medium but those of c-myc were transiently decreased in the early period by both n-6 and n-3 polyunsaturated fatty acids. The level of tumor suppressor gen p53 mRNA , however, was increased by DHA with time. It is concluded that the cytotoxicity of lipid peroxide and increased expression of tumor suppressor gene p53 are at least partly responsible for the inhibitory effect of DHA on growth of breast cancer cells.

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Nutrient dynamics in montane wetlands, emphasizing the relationship between cellulose decomposition and water chemistry

  • Kim, Jae Geun
    • 한국습지학회지
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    • 제7권4호
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    • pp.33-42
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    • 2005
  • Wetlands often function as a nutrient sink. It is well known that increased input of nutrient increases the primary productivity but it is not well understood what is the fate of produced biomass in wetland ecosystem. Water and sediment quality, decomposition rate of cellulose, and sediment accumulation rate in 11 montane marshes in northern Sierra Nevada, California were analyzed to trace the effect of nitrogen and phosphorus content in water on nutrient dynamics. Concentrations of ammonium, nitrate, soluble reactive phosphorus (SRP) in water were in the range of 27 to 607, 8 to 73, and 6 to 109 ppb, respectively. Concentrations of ammonium, calcium, magnesium, sodium, and potassium in water were the highest in Markleeville, which has been impacted by animal farming. Nitrate and SRP concentrations in water were the highest in Snow Creek, which has been impacted by human residence and a golf course. Cellulose decomposition rates ranged from 4 to 75 % per 90 days and the highest values were measured in Snow Creek. Concentrations of total carbon, nitrogen, and phosphorus in sediment ranged from 8.0 to 42.8, 0.5 to 3.0, and 0.076 to 0.162 %, respectively. Accumulation rates of carbon, nitrogen, and phosphorus fluctuated between 32.7 to 97.1, 2.4 to 9.0, and 0.08 to $1.14gm^{-2}yr{-1}$, respectively. Accumulation rates of carbon and nitrogen were highest in Markleeville and that of phosphorus was highest in Lake Van Norden. Correlation analysis showed that decay rate is correlated with ammonium, nitrate, and SRP in water. There was no correlation between element content in sediment and water quality. Nitrogen accumulation rate was correlated with ammonium in water. These results showed that element accumulation rates in montane wetland ecosystems are determined by decomposition rate rather than nutrient input. This study stresses a need for eco-physiological researches on the response of microbial community to increased nutrient input and environmental change because the microbial community is responsible for the decomposition process.

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$^1H$ NMR-Based Metabolomic Approach for Understanding the Fermentation Behaviors of Wine Yeast Strains

  • Son, Hong-Seok;Hwang, Geum-Sook;Kim, Ki-Myong;Kim, Eun-Young;Berg, Frans van den;Park, Won-Mok;Lee, Cherl-Ho;Hong, Young-Shick
    • 한국미생물학회:학술대회논문집
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    • 한국미생물학회 2009년도 International Meeting of the Microbiological Society of Korea
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    • pp.78-78
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    • 2009
  • $^1H$ NMR spectroscopy coupled with multivariate statistical analysis was used for the first time to investigate metabolic changes in musts during alcoholic fermentation and wines during ageing. Three Saccharomyces cerevisiae yeast strains (RC-212, KIV-1116 and KUBY-501) were also evaluated for their impacts on the metabolic changes in must and wine. Pattern recognition (PR) methods, including PCA, PLS-DA and OPLS-DA scores plots, showed clear differences for metabolites among musts or wines for each fermentation stage up to 6 months. Metabolites responsible for the differentiation were identified to valine, 2,3-butanediol (2,3-BD), pyruvate, succinate, proline, citrate, glycerol, malate, tartarate, glucose, N-methylnicotinic acid (NMNA), and polyphenol compounds. PCA scores plots showed continuous movements away from days 1 to 8 in all musts for all yeast strains, indicating continuous and active fermentation. During alcoholic fermentation, highest levels of 2,3-BD, succinate and glycerol were found in musts with the KIV-1116 strain, which showed the fastest fermentation or highest fermentative activity of the 3 strains, whereas the KUBY-501 strain showed the slowest fermentative activity. This study highlights the applicability of NMR-based metabolomics for monitoring wine fermentation and evaluating the fermentative characteristics of yeast strains.

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