• Title/Summary/Keyword: In vitro screening

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Arctigenin from Burdock Root Exhibits Potent Antibacterial and Anti-Virulence Properties against Pseudomonas aeruginosa

  • Abdulrahman E. Koshak;Mahmoud A. Elfaky;Hossam M. Abdallah;Dina A. I. Albadawi;Gamal A. Mohamed;Sabrin R. M. Ibrahim;Abdulrahim A. Alzain;El-Sayed Khafagy;Azza A. H. Rajab;Wael A. H. Hegazy
    • Journal of Microbiology and Biotechnology
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    • v.34 no.8
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    • pp.1642-1652
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    • 2024
  • Arctium lappa (Burdock) root is used in various culinary applications especially in Asian Cuisine. Arctigenin (ARC) is a polyphenolic compound abundant in the roots of the burdock plant from which it derives its name. The emergence of bacterial resistance is a growing global worry, specifically due to the declining availability of new antibiotics. Screening for the antibacterial candidates among the safe natural products is a promising approach. The present study was aimed to assess the antibacterial activity of ARC against Pseudomonas aeruginosa exploring its effect on the bacterial cell membrane. Furthermore, the anti-virulence activities and anti-quorum sensing (QS) activities of ARC were in vitro, in vivo and in silico assessed against P. aeruginosa. The current results showed the ARC antibacterial activity was owed to its disruption effect of the cell membrane. ARC at sub-MIC significantly decreased the formation of biofilm, motility, production of extracellular enzymes and in vivo protected mice against P. aeruginosa. These anti-virulence activities of ARC are owed to its interference with bacterial QS and its expression. Furthermore, ARC showed mild effect on mammalian erythrocytes, low probability to induce resistance and synergistically combined with antibiotics. In summary, the promising anti-virulence properties of ARC indicate its potential as an effective supplement to conventional antibiotics for treating severe P. aeruginosa infections.

Measurement of Methane Production from Ruminants

  • Bhatta, Raghavendra;Enishi, Osamu;Kurihara, Mitsunori
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.8
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    • pp.1305-1318
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    • 2007
  • On a global scale agriculture and in particular enteric fermentation in ruminants is reported to produce about one fourth (21 to 25%) of the total anthropogenic emissions of methane ($CH_4$). Methane is produced during the anaerobic fermentation of hydrolyzed dietary carbohydrates in the rumen and represents an energy loss to the host besides contributing to emissions of greenhouse gases into the environment. However, there appears to be uncertainty in the $CH_4$ estimation from livestock due to the limited availability of data to document the variability at the farm level and also due to the significant impact of diet on the enteric $CH_4$ production. The methane mitigation strategies require robust prediction of emissions from rumen. There are many methods available which would be suitable for measuring $CH_4$ produced from the various stages of animal production. However, several factors need to be considered in order to select the most appropriate technique like the cost, level of accuracy required and the scale and design of the experiments to be undertaken. Selection of any technique depends on the accuracy as each one has its advantages and disadvantages. Screening of mitigation strategies may be evaluated using individual animal before large-scale trials on groups of animals are carried out. In this review various methods for the estimation of methane production from ruminants as well as for the determination of methane production potential of ruminant feeds are discussed. The advantages and disadvantages of the methods starting from respiration chamber, ventilated hood, facemask, sulphur hexafluoride ($SF_6$) tracer technique, prediction equations and meteorological methods to in vitro methods are detailed.

An Autochthonous Case of Canine Visceral Leishmaniasis in Korea

  • Bhang, Dong Ha;Choi, Ul Soo;Kim, Hyun Jeong;Cho, Kyoung-Oh;Shin, Sung-Shik;Youn, Hee-Jeong;Hwang, Cheol-Yong;Youn, Hwa-Young
    • Parasites, Hosts and Diseases
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    • v.51 no.5
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    • pp.545-549
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    • 2013
  • A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author's knowledge this is the first autochthonous case of canine leishmaniasis in Korea.

Recently Isolated Bioactive Compounds from Korean Marine Sponges

  • Lim, Young-Ja;Kim, Jung-Sun;Chung J. Shim;Lee, Chong-O.;Im, Kwang-Sik;Jee H. Jung
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1998.11a
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    • pp.89-94
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    • 1998
  • Marine sponges are recognized as a plentiful source of diverse biologically active secondary metabolites. Recently, we have initiated a research to discover antitumor constituents from the marine sponges collected from Korean Waters. Marine sponges collected from the South Sea of Korea were screened for several biological activities including such as brine shrimp lethality and cytotoxicity. Significant brine shrimp lethality was detected in the crude extract of a two-sponge association of Poecillastra sp. and Jaspis sp. A cross-section of this sample showed two layers of morphologically distinct sponges. The thin and dirty yellow outer layer was identified as Poecillastra sp. (Pachastrellidae), the surface of which was very rough. The light-grey inner layer was identified as Jaspis sp. (Jaspidae), the surface of which was smooth. This two-sponge association appears to be consistent as these sponges were always found in associated form regardless of collection site or collection period. Investigation of the bioactive constituents monitored by brine shrimp lethality assay led to the isolation of pectenotoxin II (PTX2) and psammaplin A as causative compounds for the brine shrimp lethality. $^1$H- and $\^$13/C-nmr signals of PTX2 was fully assigned utilizing TOCSY, HETCOR, Long-range HETCOR, and Homonuclear J-resolved 2D experiments. PTX2 displayed very potent and selective cytotoxicities in the 60 cell line panel antitumor assay at the NCI. PTX2 has progressed to acute toxicity determination and in vivo antitumor assay at the NCI (Table 1). However, significant in vitro antitumor activity of PTX2 can not be affirmed in the in vivo assay.

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Molecular Cloning of Differentially Expressed Genes in First Trap Leaf of Dionaea muscipula by Fluorescent Differential Display (형광 Differential Display법에 의한 파리지옥풀 포충잎트랩 특이발현 유전자 탐색)

  • Kang, Kwon-Kyoo;Lee, Keun-Hyang;Park, Jin-Heui;Hong, Kyong-Ei
    • Journal of Plant Biotechnology
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    • v.30 no.4
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    • pp.307-313
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    • 2003
  • Fluorescent differential display (FDD) is a method for identifying differentially expressed genes in eukaryotic cells. The mRNA FDD technology works by systematic amplification of the 3' terminal regions of mRNAs. This method involve the reverse transcription using anchored primers designed to bind 5'boundary of the poly A tails, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences. The amplified cDNA subpopulations are separated by denaturing polyacrylamide electrophoresis. To identify the genes involved in the development of first trap leaf, we applied a FDD method using mRNAs from leaf base, first trap leaf and flower tissue, respectively. We screened several genes that expressed specifically in first trap leaf. Nucleotide sequence analysis of these genes revealed that these were protease inhibitor (PI), myo-inositol-1-phosphate synthase and lipocalin-type prostaglandin D synthase. Northern blot analysis showed that these genes were expressed specifically in first trap leaf (in vivo and in vitro). FDD could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Screening of immunoactive ingredients in frequently consumed food in Korea

  • Gil, Na-Young;Lee, Sang-Myeong;Mun, Ji-Young;Yeo, Soo-Hwan;Kim, So-Young
    • Journal of Biomedical and Translational Research
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    • v.19 no.4
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    • pp.92-102
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    • 2018
  • The objectives of this study were to find out the plant to enhance immune activity among 42 kinds of foods frequently consumed by the Korean elderly consisting of 5 food groups and 5 wild plants. Each sample was assessed the immunoactive effect by measuring $NF-{\kappa}B/AP1$ gene expression, nitric oxide and cytokine production in $RAW-Blue^{TM}$ cell. Soybean sprouts of 47 plants showed the highest $NF-{\kappa}B/AP1$ gene expression at the level of $1.13{\pm}0.03$ (O.D. 650 nm) and Soritae, sweet potato, banana, apple, garlic, crown daisy, cabbage and Ailanthus altissima also had high activity of $NF-{\kappa}B/AP1$ gene in $RAW-Blue^{TM}$ cell stimulated by LPS. NO production of Ailanthus altissima was significantly higher than that of other plants and 16 plants of glutinous sorghum, black rice, Seoritae, Heuktae, sweet potato, banana, apple, garlic, mungbean sprouts, spinach, crown daisy, young pumpkin, cabbage, soybean sprouts, Actinidia arguta and Aster scaber were the next best activity. The above results selected 17 out of 47 plant samples. Moreover, soybean sprouts was significantly shown to increase $TNF-{\alpha}$ ($1,509.55{\pm}1.38pg/mL$) and $IL-1{\beta}$ ($54.56{\pm}1.08pg/mL$) cytokines in comparison with RAW-Blue cell stimulated by LPS. According to the results of in vitro evaluation, the ethanol extract of soybean sprout increased the production of immune-enhancing cytokines by proliferation of macrophages. In addition, $NF-{\kappa}B$ transcription factor activity and NO production ability were excellent, and it was selected as a material having excellent immunological activity.

Cloning, Expression, and Polymerization Assay of FtsZ Protein from Staphylococcus aureus (Staphylococcus aureus FtsZ의 클로닝, 발현 및 폴리머 형성 활성 분석)

  • Son, Sang Hyeon;Lee, Dong Yun;Kim, Ye Jun;Ko, Sooho;Cho, Seong Jun;Jung, Hyo Cheol;Lee, Hyung Ho
    • Microbiology and Biotechnology Letters
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    • v.40 no.3
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    • pp.274-277
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    • 2012
  • Cytokinesis is the final stage of cell division, dividing one mother cell into two daughter cells. For the cutting of a plasma membrane during bacterial cytokinesis, a tubulin homolog FtsZ protein is recruited from the cytoplasm to the division site. FtsZ protein polymerizes in a GTP-dependent manner and its N-terminal domain has a GTPase activity. In this study, we have begun to characterize FtsZ from Staphylococcus aureus (SA). Full-length SA FtsZ was cloned into pRSFDuet-1 vector and the clone was transformed into a BL21 (DE3) star cell. The recombinant SA FtsZ protein was purified using Ni-NTA affinity chromatography and dialysis. Using a spectrofluorometer, we showed that SA FtsZ undergoes a GTP-dependant polymerization in vitro. The polymer of the SA FtsZ protein disappeared after a few minutes, suggesting that the polymer is degraded as the GTP is consumed. This assay system may well be applied for inhibitor screening targeting S. aureus FtsZ.

Screening Assay for Identification of Endocrine Disruptors with Androgen Activities using LNCaP Cells (LNCaP 세포주를 이용한 내분비계장애물질중 안드로겐성 확인시험을 위한 검색법)

  • 김진호;정혜주;김영옥;정승태;박재현;조대현;김동섭
    • Toxicological Research
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    • v.18 no.1
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    • pp.59-64
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    • 2002
  • Substantial evidences have been accumulated about the hormone-like effects of exogenous substances such as pesticides and industrial chemicals during past years. The effects of these substances on the endocrine system are believed to be either enhancing or reducing of various endocrine action. It is necessary to identify putative causal agents by the batter system and to assess their ability to disrupt the endocrine system. A variety of in vitro and In vivo approaches have been used to determine the androgenic effects of environmental chemicals. To establish the method for assessment of the putative endocrine disruptors with androgenic activity, we carried out the cell proliferation assay by MTS method after treatment with the various concentration of testosterone in LNCaP cells (human prostatic cancer cell line) and also observed the expression of androgen-related genes by quantitative RT-PCR. In the cell proliferation assay, the results showed that the grouth of LNCaP cells increased within level of at least 10pM testosterone. We measured by quantitative RT-PCR method on the effects of testosterone on mRNA expression of androgen receptor (AR), prostate-specific antigen (PSA), bone morphogenetic protein (BMP) and BMP receptor (BMPR) In LNCaP cells. The results demonstrated that mRNA expression of PSA and BMPR-IB was observed differently within level of at least 0.01 pM testosterone compared with non-treated control. These observations suggest that the detection of PSA and BMPR-IB mRNA by the quantitative RT-PCR in LNCaP cells is very sensitive method to identify the endocrine disruptors to have the androgenic effects.

In Vitro Screening for Antimicrobial Activity of Chitosans and Chitooligosaccharides, Aiming at Potential Uses in Functional Textiles

  • Fernandes, Joao C.;Tavaria, Freni K.;Fonseca, Susana C.;Ramos, Oscar S.;Pintado, Manuela E.;Malcata, F. Xavier
    • Journal of Microbiology and Biotechnology
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    • v.20 no.2
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    • pp.311-318
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    • 2010
  • Antimicrobial finishing of textiles has been found to be an economical way to prevent (or treat) skin disorders. Hence, this research effort was aimed at elucidating the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon six dermal reference microorganisms, as well as the influence of the interactions with cotton fabrics on said activity. Using 3 chitosans with different MWs, as well as two chitooligosaccharide (COS) mixtures, a relevant antimicrobial effect was observed by 24 h for the six microorganisms tested; it was apparent that the antimicrobial effect is strongly dependent on the type of target microorganism and on the MW of chitosan - being higher for lower MW in the case of E. coli, K. pneumoniae, and P. aeruginosa, and the reverse in the case of both Gram-positive bacteria. Furthermore, a strong antifungal effect was detectable upon C. albicans, resembling the action over Gram-positive bacteria. Interactions with cotton fabric resulted in a loss of COS activity when compared with cultured media, relative to the effect over Gram-negative bacteria. However, no significant differences for the efficacy of all the 5 compounds were observed by 4 h. The three chitosans possessed a higher antimicrobial activity when impregnated onto the fabric, and presented a similar effect on both Gram-positive bacteria and yeast, in either matrix. Pseudomonas aeruginosa showed to be the most resistant microorganism to all five compounds.

Identification of the novel substrates for caspase-6 in apoptosis using proteomic approaches

  • Cho, Jin Hwa;Lee, Phil Young;Son, Woo-Chan;Chi, Seung-Wook;Park, Byoung Chul;Kim, Jeong-Hoon;Park, Sung Goo
    • BMB Reports
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    • v.46 no.12
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    • pp.588-593
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    • 2013
  • Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis.