• Parasites, Hosts and Diseases
• /
• 제53권4호
• /
• pp.385-394
• /
• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• The Plant Pathology Journal
• /
• 제31권2호
• /
• pp.123-131
• /
• 2015

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least $1.47{\times}10^2copies/{\mu}l$ of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or $10^{-6}$ dilution point of 0.12 at $OD_{600}$ units of cells per reaction using a calibrated cell suspension.

• Archives of Pharmacal Research
• /
• 제28권8호
• /
• pp.923-929
• /
• 2005

Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of $11.9 {\mu}g/mL,\;9.4{\mu}g/mL,\;and\;12.9{\mu}g/mL$, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.

• 대한본초학회지
• /
• 제26권3호
• /
• pp.83-90
• /
• 2011

Objectives : This study was performed to investigate the influence of black ginseng radix extracts (BG) and ginsenoside Rg3, Rg5 on basic fibroblast growth factor (bFGF) induced proliferation, migration and capillary tubule-like formation of human umbilical vein endothelial cells (HUVECs). Methods : HUVECs were cultured with BG and ginsenoside Rg3, Rg5 at different concentrations (60, 125, 250, 500, $1,000{\mu}g/m\ell$) for 2 day In the presence of bFGF, respectively. XTT was used to detect the proliferation. Migration and tube formations were examined to detect the antiangiogenesis. Also, the chick embryo chorioallantoic membrane (CAM) assay was performed to detect the antiangiogenesis. Results : BG and ginsenoside Rg3, Rg5 significantly inhibited bFGF-induced endothelial cell proliferation and migration in a dose-dependent manner. Tube formation in bFGF-induced HUVECs were suppressed by BG and ginsenoside Rg3, Rg5. Moreover, BG and ginsenoside Rg3, Rg5 (30-$50{\mu}g$/egg) inhibited new blood vessel formation on the growing CAM. Conclusions:Based on the present results, it can be suggested that BG has a potential chemopreventive agent via antiangiogenesis.

• Molecules and Cells
• /
• 제41권9호
• /
• pp.842-852
• /
• 2018

Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to ${\beta}-trefoil$ domain (BTD) of CSL using a conserved ${\varphi}W{\varphi}P$ motif (${\varphi}$ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ${\varphi}W{\varphi}P$ sequence (APIWRP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.

• Journal of Periodontal and Implant Science
• /
• 제35권4호
• /
• pp.907-919
• /
• 2005

Porphyromonas gingivalis is a gram negative. black-pigmented anaerobe, associated with periodontitis & peri-implantitis. Fimbriae(fimA) of P. gingivalis are filamentous components on the cell surface and important in the colonization and invasion of periodontal tissue. But all P. gnigivalis strains don't have equal pathogenicity, inequality among strains originates from different fimA genotype. P. gnigivalis fimA gene encoding fimbrillin(structural subunit of fimbriae) has been classified into 5 genotypes(types I to V) based on the nucleotide sequences. In the present study, we examined the prevalence of these fimA genotypes in patients with dental implant and the relationship between prevalence of these genotypes and a condition of peri-implant tissue. Dental plaque specimens obtained from 189 peri-implant sulci of 97 patients with dental implants were analyzed by 16S rRNA fimA gene-directed PCR assay. P. gingivalis were detected in 86.2% of the alll samples. Among the P. gingivalis-positive samples, a significant difference in the occurrence of typeII was observed between test and the two control groups. In two control groups, typeII fimA were detected in 6.3%(PD<5mm/BOP-). 18.7%(PD<5mm/BOP+). In the test $group(PD{\geqq}5mm/BOP+)$, type II fimA genotype were detected most frequently in 50.0% . And a correlation between specific fimA types and peri-implantitis was found in $typeII(R^2=l.105)$. These results suggest that P. gingivalis strains that possess typeII fimA are gradually increased, as a condition of peri-implant tissue is getting complicated and are closely associated with peri-implant health status. We speculate that these organisms be involved in peri-implantitis

• Korean Chemical Engineering Research
• /
• 제53권4호
• /
• pp.472-477
• /
• 2015

본 연구에서는 패러데이 모트를 사용한 기존의 피코리터 주입용 미세유체 칩에 은 나노입자를 이용한 전극을 추가하여 전압을 낮추며 효율을 높이는 실험을 수행하였다. 먼저, 복잡한 제조공정에서 탈피하여 은 나노입자 용액을 한 방울 떨어뜨리는 간단한 과정만으로 미세유체 피코리터 주입기 내에 전극을 제조하였다. 본 개념을 통한 은 나노입자 전극과 패러데이 모트가 통합된 미세유체 칩은 은 나노입자 전극을 사용하지 않는 기존 미세유체 칩의 피코리터 주입 시작 전압인 260 V 보다 낮은 전압인 180 V에서 피코리터 주입이 작동되었다. 또한 미세유체 피코리터 주입기는 피코리터 주입 부피를 7.5 pL부터 27.5 pL까지 정밀하게 조절할 수 있음을 주된 장점으로 하고 있다. 본 미세유체 피코리터 주입기는 미세유체 시스템의 새로운 기능을 설계함으로써 각 연구분야를 탐구할 유용한 플랫폼으로 기대되고 있다.

• Bulletin of the Korean Chemical Society
• /
• 제31권12호
• /
• pp.3567-3572
• /
• 2010

Black pine barks from the southern region of Korea were extracted using pressurized hot water and the water soluble extracts were then separated in a stepwise fashion using a variety of solvents, column chromatography (CC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The antioxidant activities of each fraction and the active compounds were determined based on the radical scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH), reductive potential of ferric ion, and total phenol contents. A DPPH test showed that the half maximal effective concentration ($EC_{50}$ value : $6.59{\pm}0.31\;{\mu}g/mL$) of the ethyl acetate fraction (ca. 0.67%) was almost the same as that of the control compounds and inversely proportional to the value of the total phenol contents. The cell viability of the water extracts was confirmed by methyl thiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) with enzyme linked immune sorbent assay (ELISA). Catechin, epicatechin, quercetin and ferulic acid were isolated from the ethyl acetate fraction as active compounds and identified by nuclear magnetic resonance. The antioxidant activity as value of DPPH of each of the separated compounds was lower than the ethyl acetate fraction, and ferulic acid was the lowest among these compounds.

• Food Science and Biotechnology
• /
• 제16권1호
• /
• pp.99-103
• /
• 2007

The purpose of this study was to develop a simple detection method, possibly at the species-level, that allows for large-scale screening of bifidobacteria. Human fecal samples were plated on MRS-raffinose agar containing cysteine and neomycin sulfate, serving as selective pressure for bifidobacteria, and 0.003%(w/v) bromocresol green. All of the test strains grew well on this medium at $37{\pm}1^{\circ}C$, forming white colonies surrounded by yellow halos, which presented a sharp contrast against the green background. In this disc assay, the required incubation time to develop a yellowish zone varied with the species of Bifidobacterium that was tested, allowing for differential counts and easy identification at the species-level: 10-14 hr for B. bifidum, 20-22 hr for B. catenulatum and B. infantis. and 24-25 hr for B. longum and B. breve. No apparent color was observed for B. angulatum and B. adolescentis 28 hr after inoculation. To evaluate the results of pH indicator-based identification, individual isolates were subjected to a colony-PCR experiment with genus-specific primers. The amplified products from the isolates were in good accordance with those from the reference strains at a level of 95% agreement. These results suggest that the present method could be conveniently applied to cell counts, as well as to the preliminary identification of bifidobacteria from a variety of sample types including human feces, dairy products, and commercial probiotic supplements.

• KSBB Journal
• /
• 제22권6호
• /
• pp.393-400
• /
• 2007

현재 많은 대학과 기업에서 다양한 방법으로 상용화가 가능한 protein microarray의 개발을 위해 많은 연구를 집중하고 있다. Protein microarray의 제작 및 분석 조건을 최적화하기 위한 연구도 진행되고 있지만 protein microarray로 부터 얻은 분석 결과를 모든 연구자들이 공유하고 통합하기 위한 노력이 절실한 실정이다. 뿐만 아니라, PCR 같은 무한 확장 방법이 존재하지 않는 단백질의 특성을 고려할 때, 좀 더 실용적인 protein microarray를 많이 만들기 위해서는 수많은 단백질들과 결합할 수 있는 특이성이 높고 결합력이 강한 capture molecule들을 개발하는 것이 필수이다. 그러나 이러한 장애에도 불구하고 protein microarray는 아주 적은 시료량으로 high-throughput assay가 가능하다는 장점 때문에 현재의 생명과학의 발전 추세로 볼 때 앞으로 protein microarray가 조만간 실용화될 것이며 이의 시장성은 매우 클 것으로 기대된다. 보다 빠른 실용화를 위해서는 protein microarray의 개발에 필요한 기반 기술의 개발과 동시에 이를 활용하기 위한 contents의 개발도 절실히 요구된다.