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Effects of polygalacin D extracted from Platycodon grandiflorum on myoblast differentiation and muscle atrophy (길경에서 추출한 polygalacin D가 근원세포 분화 및 근위축에 미치는 영향)

  • Eun-Ju Song;Ji-Won Heo;Jee Hee Jang;Eonmi Kim;Yun Hee Jeong;Min Jung Kim;Sung-Eun Kim
    • Journal of Nutrition and Health
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    • v.56 no.6
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    • pp.602-614
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    • 2023
  • Purpose: The balance between synthesis and degradation of proteins plays a critical role in the maintenance of skeletal muscle mass. Mitochondrial dysfunction has been closely associated with skeletal muscle atrophy caused by aging, cancer, and chemotherapy. Polygalacin D is a saponin derivative isolated from Platycodon grandiflorum (Jacq.) A. DC. This study aimed to investigate the effects of polygalacin D on myoblast differentiation and muscle atrophy in association with mitochondrial function in in vitro and in zebrafish models in vivo. Methods: C2C12 myoblasts were cultured in differentiation media containing different concentrations of polygalacin D, followed by the immunostaining of the myotubes with myosin heavy chain (MHC). The mRNA expression of markers related to myogenesis, muscle atrophy, and mitochondrial function was determined by real-time quantitative reverse transcription polymerase chain reaction. Wild type AB* zebrafish (Danio rerio) embryos were treated with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with or without polygalacin D, and immunostained to detect slow and fast types of muscle fibers. The Tg(Xla.Eef1a1:mitoEGFP) zebrafish expressing mitochondria-targeted green fluorescent protein was used to monitor mitochondrial morphology. Results: The exposure of C2C12 myotubes to 0.1 ng/mL of polygalacin D increased the formation of MHC-positive multinucleated myotubes (≥ 8 nuclei) compared with the control. Polygalacin D significantly increased the expression of MHC isoforms (Myh1, Myh2, Myh4, and Myh7) involved in myoblast differentiation while it decreased the expression of atrophic markers including muscle RING-finger protein-1 (MuRF1), mothers against decapentaplegic homolog (Smad)2, and Smad3. In addition, polygalacin D promoted peroxisome proliferator-activated receptor-gamma coactivator (Pgc1α) expression and reduced the level of mitochondrial fission regulators such as dynamin-1-like protein (Drp1) and mitochondrial fission 1 (Fis1). In a zebrafish model of FOLFIRI-induced muscle atrophy, polygalacin D improved not only mitochondrial dysfunction but also slow and fast muscle fiber atrophy. Conclusion: These results demonstrated that polygalacin D promotes myogenesis and alleviates chemotherapy-induced muscle atrophy by improving mitochondrial function. Thus, polygalacin D could be useful as nutrition support to prevent and ameliorate muscle wasting and weakness.

Breeding a new cultivar of Pleurotus ostreatus, 'Otari' and its characteristics (느타리 신품종 '오타리'의 육성 및 특성)

  • Minji Oh;Ji-Hoon Im;Minseek Kim;Doo-Ho Choi;Eun-Ji Lee;Sung-I Woo;Youn-Lee Oh
    • Journal of Mushroom
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    • v.22 no.3
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    • pp.128-132
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    • 2024
  • Oyster mushroom is one of the most widely cultivated and consumed mushrooms in Korea, and mechanization and automation of cultivation systems have enabled mass production. Many cultivars have been developed to replace the old ones such as 'Suhan' and 'Chunchuneutari 2 ho,' which have been cultivated for over 20 years. Among these, 'Soltari' was developed in 2015. Although it has excellent quality, its cultivation is challenging and the productivity is somewhat lower. To address these issues, the Mushroom Division at the National Institute of Horticultural and Herbal Science selected the genetic resource KMCC05165 and attempted hybridization between monokaryons from KMCC05165 and 'Soltari(KMCC04940)'. Through repeated cultivation tests and evaluation of fruiting body characteristics, the superior strain 'Po-2019-smj22' was selected and finally named 'Otari'. The optimal mycelial growth temperature of 'Otari' was between 25 and 30℃ and optimal fruiting body growth temperature was between 13 and 18℃. Mycelial growth on PDA medium was best at 25℃, and at the same temperature, mycelial growth was similar across four media: PDA, MEA, MCM, and YM. In 1,100 mL bottle cultivation, the yield was approximately 174 g, which is about 5% higher than the control cultivar 'Soltari', and the number of valid individuals was also higher at about 25. The diameter and height of the pileus were 29.8 mm and 17.6 mm, respectively, slightly smaller than 'Soltari', and the stipe was thin and long with a thickness of 12.2 mm. Additionally, the pileus' lightness index (L index) was 30.7, indicating a darker brown color compared to 'Soltari.' With excellent mycelial growth, ease of cultivation, and high yield, the new cultivar 'Otari' is expected to be widely adopted by domestic oyster mushroom farms.

The Difference in Chemokine Expression in Airway Epithelial Cells According to the Virulence of Tubercle Bacilli (결핵균 독성 여부에 따른 기도 상피세포의 Chemokine 발현에 관한 연구)

  • Kwon, O-Jung;Kim, Ho-Joong;Kim, Jung-Hee;Kim, Ho-Cheol;Suh, Gee-Young;Park, Jeong-Woong;Park, Sang-Joon;Chung, Man-Pyo;Choi, Dong-Chull;Rhee, Chong-H.
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.4
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    • pp.729-741
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    • 1997
  • Background : We have recently reported that airway epithelial cells can produce RANTES and IL-8 in response to the stimulation of tubercle bacilli suggesting a certain role of airway epithelial cells in the pathogenesis of pulmonary tuberculosis. The pathogenesis of tuberculosis is determined by several factors including phagocytosis, immunological response of host, and virulence of tubercle bacilli. Interestingly, there have been reports suggesting that difference in immunological response of host according to the virulence of tubercle bacilli may be related with the pathogenesis of tuberculosis. We, therefore, studied the expressions and productions of RANTES and IL-8 in airway epithelial cells in response to tubercle bacilli(H37Rv, virulent strain and H37Ra, avirulent strain), in order to elucidate the possible pathophysiology of pulmonary tuberculosis. Methods : Peripheral blood monocytes were isolated from normal volunteers. Peripheral blood monocytes (PBM) were stimulated with LPS($10{\mu}g/ml$), H37Rv, or H37Ra($5{\times}10^5$ bacilli/well) along with normal control for 24 hours. A549 cells were stimulated with supernatants of cultured PBM for 24 hours. ELISA kit was used for the measurement of $TNF{\alpha}$ and IL-$1{\beta}$ production in supernatants of cultured PBM and for the measurement of RANTES and IL-8 in supernatants of cultured A549 cells. Northern blot analysis was used for the measurement of RANTES and IL-8 mRNA expression in cultured A549 cells. Results : $TNF{\alpha}$ and IL-$1{\beta}$ productions were increased in cultured PBM stimulated with LPS or tubercle bacilli(H37Rv or H37Ra) compared with the control. There was, however, no difference in $TNF{\alpha}$ and IL-$1{\beta}$ production between cultured PBM stimulated with H37Rv and H37Ra. RANTES and IL-8 expressions and productions were also increased in cultured A549 cells stimulated with LPS or tubercle bacilli compared with the control. RANTES and IL-8 mRNA expressions were significantly increased in cultured A549 cells stimulated with H37Ra-conditioned media(CM) compared with A549 cells stimulated with H37Rv-CM (p<0.05). However, there was no difference in RANTES and IL-8 productions between A549 cells stimulated with H37Rv-CM and H37Ra-CM. Conclusion : Airway epithelial cells can produce the potent chemokines such as RANTES and IL-8, in response to the stimulation of tubercle bacilli. These results suggest that airway epithelial cells may play a certain role in the pathogenesis of pulmonary tuberculosis. However, the role of airway epithelial cells in the pathogenesis of tuberculosis according to the virulence of tubercle bacilli was not clear in this study.

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Matrix Metalloproteinase in Idiopathic Pulmonary Fibrosis (특발성 폐섬유화증환자의 기관지폐포세척액 및 폐포대식세포 배양액의 Matrix metalloproteinase의 변화)

  • Park, Joo-Hun;Shim, Tae-Sun;Lim, Chae-Man;Koh, Youn-Suck;Lee, Sang-Do;Kim, Woo-Sung;Kim, Won-Dong;Kim, Dong-Soon
    • Tuberculosis and Respiratory Diseases
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    • v.51 no.4
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    • pp.303-314
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    • 2001
  • Background : Matrix metalioproteinase(MMP)-2 and MMP-9 have been known to play an important role in cell migration and the tissue remodeling process by type IV collagen lysis, a major component of the basement membrane. Intra-alveolar fibrosis, secondary to an injury to the basement membrane of the alveolar epithelial lining, is a major process in the pathogenesis of idiopathic pulmonary fibrosis(IPF). Therefore, MMP-2 and MMP-9 was hypothesized to play an important role in IPF pathogenesis. As a result, their level may reflect the activity or prognosis. Method : Forty one progressive IPF patients(age $59.82{\pm}1.73$ years, M:F=23:18), 16 patients with stable IPF for more than one year without therapy(age : $63.6{\pm}2.8$ years, M:F=13:3), and 7 normal controls were enrolled in this study. The MMP-2 and MMP-9 levels in the BAL fluid and alveolar macrophage conditioned media(AM-CM) were measured by zymography and the TIMP-1 level was measured by ELISA. Results : 1) The MMP-2 level in BALF was highest in the progressive IPF group ($1.36{\pm}0.28$) followed by the stable group ($0.46{\pm}0.13$) and the controls ($0.08{\pm}0.09$), which was statistically significant. The MMP-9 level of the IPF ($0.31{\pm}0.058$) and the stable group ($0.22{\pm}0.078$) were higher than that of the control group ($0.002{\pm}0.004$). In the AM-CM, only MMP-9 was detected, which was significantly higher in IPF group ($0.80{\pm}0.1O$) than in the control group($0.23{\pm}0.081$). The TIMP-1 level was also higher in both the IPF ($36.34{\pm}8.62\;{\mu}g/ml$) and stable group ($20.83{\pm}8.53\;{\mu}g/ml$) compared to the control group ($2.80{\pm}1.05\;{\mu}g/ml$) (p<0.05). 3) There was a correlation between the MMP-2 level in the BALF with the total cell number(r=0.298) and neutrophils(r=0.357) (p<0.05), and the MMP-9 level with the number of neutrophils (r=0.407) and lymphocytes (r=0.574)(p<0.05). The TIMP-1 level correlated with the total number of cell (r=0.338, p<0.05) and neutrophils(r=0.449, p=0.059). Conclusion : Both MMP and TIMP appear to play an important role in IPF pathogenesis, and their level may reflect the disease activity.

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Effects of Benzo〔a〕pyrene on Growth and Photosynthesis of Phytoplankton (식물플랑크톤의 성장과 광합성에 대한 benzo〔a〕pyrene의 영향)

  • Kim, Sun-Ju;Shin, Kyung-Soon;Moon, Chang-Ho;Park, Dong-Won;Chang, Man
    • Korean Journal of Environmental Biology
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    • v.22
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    • pp.54-62
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    • 2004
  • We examined the impacts of anthyopogenic pollutant (benzo〔a〕pyrene) on the growth and photosynthesis of five marine phytoplankton species (Skeletonema costatum, Heterosigma akashiwo, Prorocentrum dentatum, P. minimum, Aknshiwo sanguinea), which are dominant in Korean coastal water. After the 72 h exposure to benzo〔a〕pyrene, the dramatic decrease in cell numbers was observed in the range of 1 to 10 $\mu\textrm{g}$ L$^{-1}$ for S. costatum, P. minimum, P. dentatum, whereas for A. sanguinea and H. akashiwo at the low concentrations 0.1 to 1 $\mu\textrm{g}$ L$^{-1}$ . Among the 5 phytoplankton species, the highest growth inhibition concentration ($IC_{50}$/) was 6.20 $\mu\textrm{g}$ L$^{-1}$ for P. minimum, followed by 2.14 $\mu\textrm{g}$ L$^{-1}$ for P. dentatum, 1.68 $\mu\textrm{g}$ L$^{-1}$ for S. costatum, 0.74 $\mu\textrm{g}$ L$^{-1}$ for H. akashiwo, 0.10 $\mu\textrm{g}$ L$^{-1}$ for A. sanguinea. The five species exposed to the low concentration of 1 $\mu\textrm{g}$ L$^{-1}$ were recovered after transferring to new media, but the species exposed to the high concentrations of 10 and 100 $\mu\textrm{g}$ L$^{-1}$ were not recovered, with the exception of P. minimum. Those results indicate that the thecate dinoflagellate P. minimum is most tolerant to the chemical and the athecate dinoflagellate A. sanguinea is not. Geneyally, the cell-specific photosynthetic capacity of H. akashiwo exposed to the low concentrations of 0.1 and 1 $\mu\textrm{g}$ L$^{-1}$ was higher than that of the cells in the control, whereas the cells exposed to the high concentrations of 5 and 10 $\mu\textrm{g}$ L$^{-1}$ showed the negligible photosynthetic level by the first few days of the experiment. In the case of the cells exposed to the concentration of 5 $\mu\textrm{g}$ L$^{-1}$ , after 12 days of the experiment the photosynthetic capacity was increased toward the end of the experiment. This indicates that H. akashiwo may utilize the benzo〔a〕pyrene as a carton source for its growth when exposed to low concentrations. Results suggest that anthropogenic pollutants such as benzo〔a〕pyrene may have significant influence on the succession of phytoplankton species composition and the primary production in coastal marine environments.

Spatial effect on the diffusion of discount stores (대형할인점 확산에 대한 공간적 영향)

  • Joo, Young-Jin;Kim, Mi-Ae
    • Journal of Distribution Research
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    • v.15 no.4
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    • pp.61-85
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    • 2010
  • Introduction: Diffusion is process by which an innovation is communicated through certain channel overtime among the members of a social system(Rogers 1983). Bass(1969) suggested the Bass model describing diffusion process. The Bass model assumes potential adopters of innovation are influenced by mass-media and word-of-mouth from communication with previous adopters. Various expansions of the Bass model have been conducted. Some of them proposed a third factor affecting diffusion. Others proposed multinational diffusion model and it stressed interactive effect on diffusion among several countries. We add a spatial factor in the Bass model as a third communication factor. Because of situation where we can not control the interaction between markets, we need to consider that diffusion within certain market can be influenced by diffusion in contiguous market. The process that certain type of retail extends is a result that particular market can be described by the retail life cycle. Diffusion of retail has pattern following three phases of spatial diffusion: adoption of innovation happens in near the diffusion center first, spreads to the vicinity of the diffusing center and then adoption of innovation is completed in peripheral areas in saturation stage. So we expect spatial effect to be important to describe diffusion of domestic discount store. We define a spatial diffusion model using multinational diffusion model and apply it to the diffusion of discount store. Modeling: In this paper, we define a spatial diffusion model and apply it to the diffusion of discount store. To define a spatial diffusion model, we expand learning model(Kumar and Krishnan 2002) and separate diffusion process in diffusion center(market A) from diffusion process in the vicinity of the diffusing center(market B). The proposed spatial diffusion model is shown in equation (1a) and (1b). Equation (1a) is the diffusion process in diffusion center and equation (1b) is one in the vicinity of the diffusing center. $$\array{{S_{i,t}=(p_i+q_i{\frac{Y_{i,t-1}}{m_i}})(m_i-Y_{i,t-1})\;i{\in}\{1,{\cdots},I\}\;(1a)}\\{S_{j,t}=(p_j+q_j{\frac{Y_{j,t-1}}{m_i}}+{\sum\limits_{i=1}^I}{\gamma}_{ij}{\frac{Y_{i,t-1}}{m_i}})(m_j-Y_{j,t-1})\;i{\in}\{1,{\cdots},I\},\;j{\in}\{I+1,{\cdots},I+J\}\;(1b)}}$$ We rise two research questions. (1) The proposed spatial diffusion model is more effective than the Bass model to describe the diffusion of discount stores. (2) The more similar retail environment of diffusing center with that of the vicinity of the contiguous market is, the larger spatial effect of diffusing center on diffusion of the vicinity of the contiguous market is. To examine above two questions, we adopt the Bass model to estimate diffusion of discount store first. Next spatial diffusion model where spatial factor is added to the Bass model is used to estimate it. Finally by comparing Bass model with spatial diffusion model, we try to find out which model describes diffusion of discount store better. In addition, we investigate the relationship between similarity of retail environment(conceptual distance) and spatial factor impact with correlation analysis. Result and Implication: We suggest spatial diffusion model to describe diffusion of discount stores. To examine the proposed spatial diffusion model, 347 domestic discount stores are used and we divide nation into 5 districts, Seoul-Gyeongin(SG), Busan-Gyeongnam(BG), Daegu-Gyeongbuk(DG), Gwan- gju-Jeonla(GJ), Daejeon-Chungcheong(DC), and the result is shown

    . In a result of the Bass model(I), the estimates of innovation coefficient(p) and imitation coefficient(q) are 0.017 and 0.323 respectively. While the estimate of market potential is 384. A result of the Bass model(II) for each district shows the estimates of innovation coefficient(p) in SG is 0.019 and the lowest among 5 areas. This is because SG is the diffusion center. The estimates of imitation coefficient(q) in BG is 0.353 and the highest. The imitation coefficient in the vicinity of the diffusing center such as BG is higher than that in the diffusing center because much information flows through various paths more as diffusion is progressing. A result of the Bass model(II) shows the estimates of innovation coefficient(p) in SG is 0.019 and the lowest among 5 areas. This is because SG is the diffusion center. The estimates of imitation coefficient(q) in BG is 0.353 and the highest. The imitation coefficient in the vicinity of the diffusing center such as BG is higher than that in the diffusing center because much information flows through various paths more as diffusion is progressing. In a result of spatial diffusion model(IV), we can notice the changes between coefficients of the bass model and those of the spatial diffusion model. Except for GJ, the estimates of innovation and imitation coefficients in Model IV are lower than those in Model II. The changes of innovation and imitation coefficients are reflected to spatial coefficient(${\gamma}$). From spatial coefficient(${\gamma}$) we can infer that when the diffusion in the vicinity of the diffusing center occurs, the diffusion is influenced by one in the diffusing center. The difference between the Bass model(II) and the spatial diffusion model(IV) is statistically significant with the ${\chi}^2$-distributed likelihood ratio statistic is 16.598(p=0.0023). Which implies that the spatial diffusion model is more effective than the Bass model to describe diffusion of discount stores. So the research question (1) is supported. In addition, we found that there are statistically significant relationship between similarity of retail environment and spatial effect by using correlation analysis. So the research question (2) is also supported.

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  • The Effect of Nitric Oxide Donor or Nitric Oxide Synthase Inhibitor on Oxidant Injury to Cultured Rat Lung Microvascular Endothelial Cells (산화질소 공여물과 산화질소 합성효소 길항제가 백서 폐미세혈관 내피세포 산화제 손상에 미치는 영향)

    • Chang, Joon;Michael, John R.;Kim, Se-Kyu;Kim, Sung-Kyu;Lee, Won-Young;Kang, Kyung-Ho;Yoo, Se-Hwa;Chae, Yang-Seok
      • Tuberculosis and Respiratory Diseases
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      • v.45 no.6
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      • pp.1265-1276
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      • 1998
    • Background : Nitric oxide(NO) is an endogenously produced free radical that plays an important role in regulating vascular tone, inhibition of platelet aggregation and white blood cell adhesion to endothelial cells, and host defense against infection. The highly reactive nature of NO with oxygen radicals suggests that it may either promote or reduce oxidant-induced cell injury in several biological pathways. Oxidant injury and interactions between pulmonary vascular endothelium and leukocytes are important in the pathogenesis of acute lung injury, including acute respiratory distress syndrome(ARDS). In ARDS, therapeutic administration of NO is a clinical condition providing exogenous NO in oxidant-induced endothelial injury. The role of exogenous NO from NO donor or the suppression of endogenous NO production was evaluated in oxidant-induced endothelial injury. Method : The oxidant injury in cultured rat lung microvascular endothelial cells(RLMVC) was induced by hydrogen peroxide generated from glucose oxidase(GO). Cell injury was evaluated by $^{51}$chromium($^{51}Cr$) release technique. NO donor, such as S-nitroso-N-acetylpenicillamine(SNAP) or sodium nitroprusside(SNP), was added to the endothelial cells as a source of exogenous NO. Endogenous production of NO was suppressed with N-monomethyl-L-arginine(L-NMMA) which is an NO synthase inhibitor. L-NMMA was also used in increased endogenous NO production induced by combined stimulation with interferon-$\gamma$(INF-$\gamma$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), and lipopolysaccharide(LPS). NO generation from NO donor or from the endothelial cells was evaluated by measuring nitrite concentration. Result : $^{51}Cr$ release was $8.7{\pm}0.5%$ in GO 5 mU/ml, $14.4{\pm}2.9%$ in GO 10 mU/ml, $32.3{\pm}2.9%$ in GO 15 mU/ml, $55.5{\pm}0.3%$ in GO 20 mU/ml and $67.8{\pm}0.9%$ in GO 30 mU/ml ; it was significantly increased in GO 15 mU/ml or higher concentrations when compared with $9.6{\pm}0.7%$ in control(p < 0.05; n=6). L-NMMA(0.5 mM) did not affect the $^{51}Cr$ release by GO. Nitrite concentration was increased to $3.9{\pm}0.3\;{\mu}M$ in culture media of RLMVC treated with INF-$\gamma$ (500 U/ml), TNF-$\alpha$(150 U/ml) and LPS($1\;{\mu}g/ml$) for 24 hours ; it was significantly suppressed by the addition of L-NMMA. The presence of L-NMMA did not affect $^{51}Cr$ release induced by GO in RLMVC pretreated with INF-$\gamma$, TNF-$\alpha$ and LPS. The increase of $^{51}Cr$ release with GO(20 mU/ml) was prevented completely by adding 100 ${\mu}M$ SNAP. But the add of SNP, potassium ferrocyanate or potassium ferricyanate did not protect the oxidant injury. Nitrite accumulation was $23{\pm}1.0\;{\mu}M$ from 100 ${\mu}M$ SNAP at 4 hours in phenol red free Hanks' balanced salt solution. But nitrite was not detectable from SNP upto 1 mM The presence of SNAP did not affect the time dependent generation of hydrogen peroxide by GO in phenol red free Hanks' balanced salt solution. Conclusion : Hydrogen peroxide generated by GO causes oxidant injury in RLMVC. Exogenous NO from NO donor prevents oxidant injury, and the protective effect may be related to the ability to release NO. These results suggest that the exogenous NO may be protective on oxidant injury to the endothelium.

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    Studies on Sclerotium rolfsii Sacc. isolated from Magnolia kobus DC. in Korea (목련(Magnolia kobus DC.)에서 분리한 흰비단병균(Sclerotium rolfsii Sacc.)에 관한 연구)

    • Kim Kichung
      • Korean journal of applied entomology
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      • v.13 no.3 s.20
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      • pp.105-133
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      • 1974
    • The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp. and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate (methyl-(butylcarbamoy 1)-2-benzimidazole carbamate), Tachigaren (3-hydroxy-5-methylisoxazole) and other chemicals were also clarified. The results are summarizee as follows: 1. There were two biologic strains, Type-l and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l being more virulent. 2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/l of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not. 3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while rnaltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results. 4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon Sources were applied but decreased with the increase of nitrogen above 0.5g/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for maximun growth of mycelia. At a higher concentration of more than 20r/l, however, mycelial growth decreased as the concentration increased, and was inhibited at l50r/l to such a degree of thiamine-free. 6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, and 16r/l in asparagine for the production of sclerotia. 7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2. 8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine+biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited. Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone. 9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ml/50ml basal medium solution. 10. $NH_4NO_3$. as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate. 11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed. and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction. 12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin. 13. Seven amino acids-aspartic acid, cystine, glysine, histidine, Iycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids. 14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied .alone and when combined with other amino acids in some cases. 15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ germinability, respectively. 17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l and type-2 were killed when dipped into the $0.05\%$ solution of mercury chloride for 180 mins. and 240 mins. respectively: and in the $0.1\%$ solution, Type-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the$0.125\%$solution. Dipping into the $5\%$ copper sulphate solution or $0.2\%$ solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-l or Type-2. 19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by $66\%$ at 0.5ppm and by $92\%$ at 2.0ppm of Benlate, and by$54\%$ at 1ppm and about $77\%$ at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlate ant at 500ppm or 1,000ppm of Tachigaren. 20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the ontrol. 21. The amount of mycelia produced by consuming 1mg of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time. 22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/g sand of Benlate or Tachigaren. 23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigaren for 10 minutes or even 20 minutes.

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