• Title/Summary/Keyword: in vitro and in vitro Screening

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Screening of Natural Products for Endothelial and Renal Nitric Oxide Production

  • Kim, Hyeyoung;Han, Sang-Won
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1997.04a
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    • pp.90-90
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    • 1997
  • Natural products, which have been used for the treatment of hypertension, diuresis and nephritis in traditional oriental medicine, were selected for the screening of nitric oxide (NO) production in endothelial cells and kidney tissues in vitro as well as in vivo by measuring the conversion of [$\^$14/C]-L-arginine to [$\^$14/C]-L-citrulline, a coproduct of the enzyme reaction with NO. Confluent monolayer of endothelial cells were used for the screening of 16 natural products. Among the natural products, Zizyphus jujuba and Codonopsis pilosula stimulated endothelial NO synthase activity. Thus, both confluent monolayer of endothelial cells and kidney homogenates (glomeruli, cortical tubules, meudllae) were treated with Zizyphus jujuba and Codonopsis pilosula (final concentration 10 $\mu\textrm{g}$/$m\ell$) and NO releases were compared with those by receptor - dependent agonists, bradykinin and ADP and receptor - independent calcium ionophore A23187 in vitro. In rat experiment, NO releases in glomeruli, cortical tubules and medullae and plasma renin activity were assessed after intraperitoneal injection of Zizyphus jujuba and Codonopsis pilosula (10 mg/kg/day for 4 days). As a result, both Zizyphus jujuba and Codonopsis pilosula significantly increased NO releases in cultured endothelial cells, kidney tissues in vitro as well as in vivo. Stimulation of NO releases by Zizyphus jujuba and Codonopsis pilosula was similar to those by receptor - dependent agonists, bradykinin and ADP and receptor - independent calcium ionophore A23187 in cultured endothelial cells. However, plasma renin activity was not influenced by these two natural products. In conclusion, stimulatory effects of Zizyphus jujuba and Codonopsis pilosula on NO release in kidney may contribute their hypotensive effects and antinephritic action possibly by increasing renal blood flow.

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Development of an Efficient In Vitro Screening Method for Selection of Resistant Lily Cultivars Against Fusarium oxysporum f. sp. lilii (백합 구근부패병 저항성 검정을 위한 기내 간편 검정법 개발)

  • Jang, Ji-Young;Moon, Ki-Beom;Ha, Jang-Ho;Park, Ji-Sun;Kim, Mi-Jin;Jeon, Jae-Heung;Lee, Geung-Joo;Kim, Hyun-Soon
    • Horticultural Science & Technology
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    • v.33 no.6
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    • pp.883-890
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    • 2015
  • The soilborne fungus Fusarium oxysporum f. sp. lilii (Fol) is a serious threat to all lily cultivars, especially infecting bulbs and flowers. It has become increasingly important to develop varieties resistant against the bulb rot disease. Genetic diversity of cultivars and reliable screening methods are required for this purpose. Here, an efficient in vitro screening system for evaluating resistance to Fol in 38 in vitro-grown lily plants was investigated. Various factors including culture conditions of Fol, inoculum density, appropriate plant materials, inoculation method and duration, and incubation period of plant materials after inoculation were combined to optimize the screening method. As a result, we suggest optimal conditions for an in vitro screening system for the selection of Fol-resistant lily cultivars as follows. Fol was grown on potato dextrose agar (PDA) medium for 6 days at $25^{\circ}C$ in darkness and used as working inoculation. Spore suspensions were prepared (inoculum density: $1.0{\times}10^4$ $spores{\cdot}mL^{-1}$), and then leaf segments $1.5{\times}2.0cm^2$ were inoculated by dipping for 22 hours at $25^{\circ}C$ in dark. Later, leaves were cultured on 0.6% agar plates at $25^{\circ}C$ and 50% humidity with a photoperiod of 16 hours light/8 hours dark (fluence rate of $40{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$) to examine the progress of bulb rot. After 7 days, disease levels were classified into indices 1 (no symptom) to 6 (serious bulb rot). Soil inoculation of Fol carried out with resistant or susceptible lily cultivars that had been selected through in vitro screening confirmed the reproducibility of results. Therefore, the in vitro screening method established in this study is efficient and reliable for selection of lily cultivars resistant against bulb rot disease.

Development of New Antitumor Drugs from Natural Sources , with Guida (항종양활성 Screening을 지표로 한 천연물의약품의 개발연구와 그 생약소재의 품질평가에 대하여(抗腫瘍活性スクリ-ニングを指標とした天然物醫藥品の開發硏究とその生約素材の品質評價について))

  • Takeya, Koichi
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 1993.08a
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    • pp.14-20
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    • 1993
  • We in anticancer drug development from natural resources have conceived and used a wide variety of experimental screening systems to support our efforts during the past 20 tears. Screens have been devided to address targets at the molecular, biochemical and cellular levels, both in vivo and in vitro. Screens have been essential for the experimental evaluation of the products from natural sources. In this congress, antitumor screening methods for deveol[ment of new drugs from natural sources and evaluation of their crude drugs are discussed.

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In Vitro Wheat Immature Spike Culture Screening Identified Fusarium Head Blight Resistance in Wheat Spike Cultured Derived Variants and in the Progeny of Their Crosses with an Elite Cultivar

  • Huang, Chen;Gangola, Manu P.;Kutcher, H. Randy;Hucl, Pierre;Ganeshan, Seedhabadee;Chibbar, Ravindra N.
    • The Plant Pathology Journal
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    • v.36 no.6
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    • pp.558-569
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    • 2020
  • Fusarium head blight (FHB) is a devastating fungal disease of wheat (Triticum aestivum L.). The lack of genetic resources with stable FHB resistance combined with a reliable and rapid screening method to evaluate FHB resistance is a major limitation to the development of FHB resistant wheat germplasm. The present study utilized an immature wheat spike culture method to screen wheat spike culture derived variants (SCDV) for FHB resistance. Mycotoxin concentrations determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) correlated significantly (P < 0.01) with FHB severity and disease progression during in vitro spike culture. Selected SCDV lines assessed for FHB resistance in a Fusarium field disease nursery in Carman, Manitoba, Canada in 2016 showed significant (P < 0.01) correlation of disease severity to the in vitro spike culture screening method. Selected resistant SCDV lines were also crossed with an elite cv. CDC Hughes and the progeny of F2 and BC1F2 were screened by high resolution melt curve (HRM) analyses for the wheat UDP-glucosyl transferase gene (TaUGT-3B) single nucleotide polymorphism to identify resistant (T-allele) and susceptible (G-allele) markers. The progeny from the crosses were also screened for FHB severity using the immature spike culture method and identified resistant progeny grouped according to the HRM genotyping data. The results demonstrate a reliable approach using the immature spike culture to screen for FHB resistance in progeny of crosses in early stage of breeding programs.

In vitro Screening of Dietary Factors on Buckwheat(Fagopyrum esculentum Moench) Influencing the Regulation of Blood Pressure, Glucose and Cholesterol Level (혈압, 혈당 및 콜레스테롤농도 조절에 영향을 주는 식이 메밀인자의 in vitro 검정)

  • 최용순;김복란;김려화;이병훈;심태흠;이상영
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.29 no.2
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    • pp.280-287
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    • 2000
  • Generally, buckwheat has been regarded as a crop of secondary importance in many countries. In vitro functionalities of buckwheats as a food were evaluated in this study. Five of buckwheat cultivars were extracted with methanol, and the extractant were dried and lyophilized, separately. Or water soluble buckwheat components were digested with the commercial enzymes and the obtained protein hydrolysate was again fractionated by acid precipitation. The antioxidant capacity of the methanol extracts determined using Fe2+-ascorbic acid system was dependent ont the cultivars: The extract of Suwon 4 showed 3.3 times stronger activity than ascorbic acid in terms of IC50. Also, the extracts of buckwheats inhibited efficiently the activities of $\alpha$-amylase and lens aldose reductase. Buckwheat soluble protein or rutin suppressed the in vitro activities of angiotensin-converting enzyme, and the inhibitory degree depended largely on the cultivars. Buckwheat proteins exerted higher hydrophobicity being related to the sterol binding capacity than casein. The results suggested that buckwheat seeds may be desirable and functional food resources in human living in current society.

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Effects of paternal age on human embryo development in in vitro fertilization with preimplantation genetic screening

  • Kim, Min Kyoung;Park, Jae Kyun;Jeon, Yunmi;Seok, Su Hee;Chang, Eun Mi;Lee, Woo Sik
    • Clinical and Experimental Reproductive Medicine
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    • v.46 no.1
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    • pp.22-29
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    • 2019
  • Objective: As paternal age increases, the quality of sperm decreases due to increased DNA fragmentation and aneuploidy. Higher levels of structural chromosomal aberrations in the gametes ultimately decrease both the morphologic quality of embryos and the pregnancy rate. In this study, we investigated whether paternal age affected the euploidy rate. Methods: This study was performed using the medical records of patients who underwent in vitro fertilization (IVF) procedures with preimplantation genetic screening (PGS) from January 2016 to August 2017 at a single center. Based on their morphological grade, embryos were categorized as good- or poor-quality blastocysts. The effects of paternal age were elucidated by adjusting for maternal age. Results: Among the 571 total blastocysts, 219 euploid blastocysts were analyzed by PGS (38.4%). When the study population was divided into four groups according to both maternal and paternal age, significant differences were only noted between groups that differed by maternal age (group 1 vs. 3, p= 0.031; group 2 vs. 4, p= 0.027). Further analysis revealed no significant differences in the euploidy rate among the groups according to the morphological grade of the embryos. Conclusion: Paternal age did not have a significant impact on euploidy rates when PGS was performed. An additional study with a larger sample size is needed to clarify the effects of advanced paternal age on IVF outcomes.

Gene functional analysis of Harmonia axyridis by in vitro transcription

  • Park, Sang-Eun;Youn, Young-Nam
    • Korean Journal of Agricultural Science
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    • v.46 no.3
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    • pp.471-488
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    • 2019
  • Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.

Discovering the anti-cancer phytochemical rutin against breast cancer through the methodical platform based on traditional medicinal knowledge

  • Jungwhoi Lee;Jungsul Lee;WooGwang Sim;Jae-Hoon Kim;Chulhee Choi;Jongwook Jeon
    • BMB Reports
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    • v.56 no.11
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    • pp.594-599
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    • 2023
  • A number of therapeutic drugs have been developed from functional chemicals found in plants. Knowledge of plants used for medicinal purposes has historically been transmitted by word of mouth or through literature. The aim of the present study is to provide a systemic platform for the development of lead compounds against breast cancer based on a traditional medical text. To verify our systematic approach, integrating processes consisted of text mining of traditional medical texts, 3-D virtual docking screening, and in vitro and in vivo experimental validations were demonstrated. Our text analysis system identified rutin as a specific phytochemical traditionally used for cancer treatment. 3-D virtual screening predicted that rutin could block EGFR signaling. Thus, we validated significant anti-cancer effects of rutin against breast cancer cells through blockade of EGFR signaling pathway in vitro. We also demonstrated in vivo anti-cancer effects of rutin using the breast cancer recurrence in vivo models. In summary, our innovative approach might be proper for discovering new phytochemical lead compounds designing for blockade of malignant neoplasm including breast cancer.

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Control of Red Pepper Anthracnose Using Bacillus subtilis YGB36, a Plant Growth Promoting Rhizobacterium (식물생장촉진근권세균 Bacillus subtilis YGB36을 이용한 고추 탄저병의 생물학적 방제)

  • Lee, Yong Yoon;Lee, Younmi;Kim, Young Soo;Kim, Hyun Sup;Jeon, Yongho
    • Research in Plant Disease
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    • v.26 no.1
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    • pp.8-18
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    • 2020
  • Red pepper, one of the major economic crops in Korea, is being affected by anthracnose disease caused by Colletotrichum acutatum. To control this disease, an antagonistic bacterial strain, Bacillus subtilis YGB36 identified by 16S rDNA sequencing, physiological and biochemical analyses is used as a biological control agent. In vitro screening revealed that the strain YGB36 possess strong antifungal activity against the pathogen Cylindrocarpon destructans. The strain exhibited cellulase, protease, amylase, siderophore production and phosphate solubility. In vitro conidial germination of C. acutatum was most drastically inhibited by YGB36 cell suspensions (106 cfu/ml) or culture filtrate. Development of anthracnose symptoms was reduced on detached immature green pepper fruits by treatment with cell suspensions, and its control value was recorded as 65.7%. The YGB36 bacterial suspension treatment enhanced the germination rate of red pepper seeds and promoted root development and growth under greenhouse conditions. The in vitro screening of fungicide and insecticide sensitivity test against YGB36 revealed that the bacterial growth was not affected by any of the insecticides, and 11 fungicides out of 21 used. Collectively, our results clearly suggest that the strain YGB36 is considered as one of the potential biocontrol agents against anthracnose disease in red pepper.

Fermentation Characteristics and Microbial Protein Synthesis in an In Vitro System Using Cassava, Rice Straw and Dried Ruzi Grass as Substrates

  • Sommart, K.;Parker, D.S.;Rowlinson, P.;Wanapat, M.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.8
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    • pp.1084-1093
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    • 2000
  • An in vitro gas production system was used to investigate the influence of various substrate mixtures on a natural mix of rumen microbes by measurement of fermentation end-products. The treatments were combinations of cassava (15.0, 30.0 and 45.0%) with different roughage sources (ruzi grass, rice straw or urea treated rice straw). Microbial biomass, net $^{15}N$ incorporation into cells, volatile fatty acid production, gas volume and rate of gas production increased linearly with increasing levels of cassava inclusion. There was also an effect of roughage source, with rice straw being associated with the lowest values for most parameters whilst similar values were obtained for ruzi grass and urea treated rice straw. The results suggest that microbial growth and fermentation rate increase as a function of readily available carbohydrate in the substrate mixture. A strong linear relationship between $^{15}N$ enrichment, total volatile fatty acid production and gas production kinetics support the suggestion of the use of the in vitro gas production system as a tool for screening feedstuffs as an initial stage of feed evaluation.