• The Korean Journal of Physiology and Pharmacology
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• 제27권3호
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• pp.267-275
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• 2023

Cardiotoxicity, particularly drug-induced Torsades de Pointes (TdP), is a concern in drug safety assessment. The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) has become an attractive human-based platform for predicting cardiotoxicity. Moreover, electrophysiological assessment of multiple cardiac ion channel blocks is emerging as an important parameter to recapitulate proarrhythmic cardiotoxicity. Therefore, we aimed to establish a novel in vitro multiple cardiac ion channel screening-based method using human iPSC-CMs to predict the drug-induced arrhythmogenic risk. To explain the cellular mechanisms underlying the cardiotoxicity of three representative TdP high- (sotalol), intermediate- (chlorpromazine), and low-risk (mexiletine) drugs, and their effects on the cardiac action potential (AP) waveform and voltage-gated ion channels were explored using human iPSC-CMs. In a proof-of-principle experiment, we investigated the effects of cardioactive channel inhibitors on the electrophysiological profile of human iPSC-CMs before evaluating the cardiotoxicity of these drugs. In human iPSC-CMs, sotalol prolonged the AP duration and reduced the total amplitude (TA) via selective inhibition of I_Kr and I_Na currents, which are associated with an increased risk of ventricular tachycardia TdP. In contrast, chlorpromazine did not affect the TA; however, it slightly increased AP duration via balanced inhibition of I_Kr and I_Ca currents. Moreover, mexiletine did not affect the TA, yet slightly reduced the AP duration via dominant inhibition of I_Ca currents, which are associated with a decreased risk of ventricular tachycardia TdP. Based on these results, we suggest that human iPSC-CMs can be extended to other preclinical protocols and can supplement drug safety assessments.

• Tuberculosis and Respiratory Diseases
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• 제44권3호
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• pp.547-558
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• 1997

연구배경 : 종양괴사인자(tumor necrosis factor ; TNF)는 다양한 생물학적 기능을 가지고 있는바, 그 중 생체외에서 증명된 뚜렷한 항암효과로 말미암아 최근 항암유전자요법의 중요한 대상으로 관심을 모으고 있다. 그러나 유전자 이입의 기술적 문제로 생체외에서 암세포에 유전자 이입을 시행한 후 이를 다시 환자의 생체내로 이식하는 방법이 연구의 주종을 이루고 있다. 그러나 저지들의 과거의 연구를 포함한 여러 연구에서 TNF가 이입된 암세포는 TNF에 대해 내성을 보이는 것으로 증명되었다. 이 획득내성의 기전을 밝히는 것이 종양생물학의 이해를 넓히고 보다 효과적인 항암유전자 요법을 개발하기 위한 매우 중요한 과제로 생각된다. 저자들은 TNF 유전자 이입에 따른 암세포의 TNF에 대한 획득 내성에 새로운 방어단백질의 합성이 관여하는 지를 규명하고자 본 실험을 수행하였다. 방 법 : TNF에 감수성을 보이는 생쥐 섬유육종 세포주인 WEHI164에 TNF-$\alpha$ 유전자를 retroviral vector를 이용하여 이입하고 TNF의 발현을 시도하여 PCR, ELISA, MTT assay로 확인하였고, TNF 유전자가 이입된 세포(WEHI164-TNF)는 TNF에 내성을 보이는지 역시 MTT assay로 검증하였다. WEHI164-TNF세포를 transcription 억제제인 actinomycin D와 translation 억제제인 cycloheximide로 처리한 후 역시 MTT assay로 TNF에 대한 감수성에 변화를 보이는지 확인하였다. 결 과 : 1) TNF-$\alpha$ 유전자 이업 및 발현 확인 PCR을 시행한 결과 TNF 유전자가 이입된 WEHI164-TNF 세포주는 790 base pair 크기의 진한 DNA band를 보인 반면 모세포주는 보이지 않아서 retroviral vector를 이용한 유전자 이입이 DNA 수준에서 이루어졌음을 확인할 수 있었다. 그리고 WEHI164-TNF의 배양상층액에서 TNF양을 ELISA와 MTT assay로 측정한 결과, 생물학적 활성을 지닌 TNF를 $10.9{\pm}1.47ng/24hr/10^6cells$ 생산함을 알 수 있었다. 2) TNF 유전자 이입 전후, 암세포의 TNF에 대한 감수성 비교 TNF 농도 100ng/ml 에서 모세포는 $73{\pm}5%$의 세포독성을 보인 반면 WEHI164-TNF 세포는 $3{\pm}2%$의 세포독성을 보여 통계적으로 유의하게 (p < 0.00) TNF에 대한 내성을 획득함을 알 수 있었다. 3) TNF 유전자 이입 후 획득된 TNF에 대한 내성의 기전 WEHI164-TNF 세포를 actinomycin D로 처리한 경우 TNF 농도 10ng/ml과 100ng/ml에서 각각 $24{\pm}7%$, $44{\pm}6%$의 세포독성을 보여 control의 $6{\pm}2%$, $17{\pm}2%$보다 통계적으로 유의하게(p < 0.01) TNF에 대한 감수성이 부분적으로 회복됨을 관찰할 수 있었다. 그러나 cycloheximide로 처리한 경우에서는 TNF에 대한 감수성에 변화를 관찰할 수 없었다. 결 론 : TNF에 감수성을 보이는 암세포주인 WEHI164에 TNF 유전자를 이입하여 TNF를 발현하게 하였을 때 그 세포 자신은 TNF에 대해 내성을 획득하게되며 이에는 미지의 방어단백질의 합성이 일부 관여할 것으로 판단된다.

• 분석과학
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• 제30권3호
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• pp.113-121
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• 2017

Now a day's rise of new antibiotic resistant bacterial strains is a global threat. Ethnic people of India have been employing Aphanamixis polystachya (Wall.) R. Parker wood extract in healing cancerous wounds. The aim of this study was to evaluate the antimicrobial activity and to identify the medicinally potent chemicals in the essential oil extract of A. polystachya. The antibacterial properties of various organic extracts were evaluated against a range of bacteria (gram-positive and gram-negative bacteria) based on the disc diffusion method and GC-MS based analysis for finding active oil extract components. All extracts of A. polystachya leaves showed potential antibacterial activity, notably ethyl acetate, while petroleum ether extracts revealed highly sensitive activity against all tested bacteria (zones of inhibition ranging from 8.83 to 11.23 mm). In addition, the petroleum ether extract had the lowest MIC value (32 to $256{\mu}g/mL$) against E. coli, S. lutea, X. campestris, and B. subtilis bacteria. The major compounds detected in oil [${\beta}$-elemene (16.04 %), ${\beta}$-eudesmol (12.78 %), ${\beta}$-caryophyllene (19.37 %), ${\beta}$-selinene (11.32 %), elemol (5.76 %), and ${\alpha}$-humulene (5.68 %)] are expected to be responsible for the potent antimicrobial activity. The results of this study offer valuable insights into the potent role of A. polystachya essential oil extract in pharmaceutical and antibiotic research.

• Restorative Dentistry and Endodontics
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• 제41권3호
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• pp.189-195
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• 2016

Objectives: A variety of root canal sealers were recently launched to the market. This study evaluated physicochemical properties, biocompatibility, and sealing ability of a newly launched resin-based sealer (Dia-Proseal, Diadent) compared to the existing root canal sealers (AHplus, Dentsply DeTrey and ADseal, Metabiomed). Materials and Methods: The physicochemical properties of the tested sealers including pH, solubility, dimensional change, and radiopacity were evaluated. Biocompatibility was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For microleakage test, single-rooted teeth were instrumented, and obturated with gutta-percha and one of the sealers (n = 10). After immersion in 1% methylene blue solution for 2 weeks, the specimens were split longitudinally. Then, the maximum length of staining was measured. Statistical analysis was performed by one-way analysis of variance followed by Tukey test (p = 0.05). Results: Dia-Proseal showed the highest pH value among the tested sealers (p < 0.05). ADseal showed higher dimensional change compared to AHplus and Dia-Proseal (p < 0.05). The solubility values of AHplus and Dia-Proseal were similar, whereas ADseal had the lowest solubility value (p < 0.05). The flow values of sealer in increasing order were AHplus, DiaProseal, and ADseal (p < 0.05). The radiopacity of AHplus was higher than those of ADseal and Dia-Proseal (p < 0.05). The cell viability of the tested materials was statistically similar throughout the experimental period. There were no significant differences in microleakage values among the tested samples. Conclusions: The present study indicates that Dia-Proseal has acceptable physicochemical properties, biocompatibility, and sealing ability.

• 대한수의학회지
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• 제29권3호
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• pp.383-391
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• 1989

This study was carried out to diagnostic test for bovine mastitis by the determination of adenosine triphosphate (ATP) based on firefly bioluminescence. The results obtained are follow; 1. The infection rate of bovine mastitis investigated with 521 cows in 47 dairy farms were found to be 3.6% of clinical form and 44.1% of subclinical form according to the degree of infection. 2. The light yield produced in firefly bioluminescence system was proportional to the concentration of ATP giving stright line within the range of 100PM~1uM. 3. When the number of somatic cell in milk was determined by the ATP assay and compared with three conventional methods such Fossomatic, California mastatic test (CMT), and rolling ball viscometer (RBV), it was shown that r=0.92 for Fossomatic, 0.63 for CMT and 0.7 for RBV. 4. The microorganisms causing mastitis were isolated Staphylococcus sp. (53.3%), Streptococcus sp. (17.9%), Micrococcus sp. (13.5%), Gram negative bacilli (6.3%), Gram positive bacilli (5.5%) and Yeast-like fungi (5.4%). 5. The endogeneous ATP levels of bacteria in a raw milk determined by the firefly bioluminescence system and compared with the results of the conventional methods. The correlation was 0.88 for raw milk.

• 대한수의학회지
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• 제58권3호
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• pp.119-124
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• 2018

This study was conducted to compare the hemostatic efficacy of three ferric subsulfate- and chitosan-based styptics as a powder and a gel containing ferric subsulfate and chitosan (FSC-PO and FSC-G, respectively) and a soaked pad containing ferric subsulfate and lidocaine (FSL-SP) using a rat tail bleeding model. The cytotoxicity of the styptics against L-929 mouse fibroblasts was also evaluated using a cell counting kit-8 assay. Four groups of 10 rats each were assigned to the three different styptics and a non-treated control groups. Rat tail tips were transected, after which styptics were applied with pressure. The wounds were observed for hemostasis for 3 min, then irrigated with saline to check for recurrent hemorrhage. L-929 mouse fibroblasts were exposed to extracts of the styptics (100 mg/mL) and their dilutions (1:10, 1:100, and 1:1,000). FSC-PO and FSC-G more effectively controlled initial hemorrhage than FSL-SP (p = 0.033). Additionally, FSC-PO and FSC-G more effectively maintained hemostasis than the control group (p = 0.02 and p < 0.01, respectively). However, all styptics showed enhanced cytotoxicity against L-929 cells in a dose-dependent manner. Therefore, although FSC-PO and FSC-G would be recommended to control hemorrhage, the benefits of styptics must be balanced against the clinical significance of their cytotoxicity.

• 대한약침학회지
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• 제13권3호
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• pp.63-71
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• 2010

Objectives: Gallic acid (GA) is the major component of tannin which could be easily founded in various natural materials such as green tea, red tea, grape juice, and Corni Fructus. The purpose of this study is to investigate the effect of Gallic acid (GA) on production of interleukin (IL) in mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharide (LPS). Methods: Productions of interleukins were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$ (multi-analyte profiling beads) technology. Firstly, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, it was incubated with the antibody-conjugated beads for 30 minutes. And detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System and concentration of interleukin was determined. Results: The results of the experiment are as follows. 1. GA significantly inhibited the production of IL-3, IL-10, IL-12p40, and IL-17 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05). 2. GA significantly inhibited the production of IL-6 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05). 3. GA diminished the production of some cytokine such as IL-4, IL-5, and IL-13 in LPS-induced mouse macrophage RAW 264.7 cells. 4. GA did not show the inhibitory effect on the production of IL-$1{\alpha}$ and IL-9 in LPS-induced mouse macrophage RAW 264.7 cells. Conclusions: These results suggest that GA has anti-inflammatory activity related with its inhibitory effects on the production of interleukins such as IL-3, IL-10, IL-12p40, IL-17, and IL-6 in LPS-induced macrophages.

• Nutrition Research and Practice
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• 제9권4호
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• pp.343-349
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• 2015

BACKGROUND/OBJECTIVES: Fermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated. MATERIALS/METHODS: Mucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells. RESULTS: Treatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Based on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate potentially stimulates the MAPK signaling pathway in intestinal cells, which is positively correlated with gut defense.

• 대한수의학회지
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• 제36권1호
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• pp.119-129
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• 1996

The present study was conducted to rapidly detect Listeria monocytogenes in raw milk. Specificity and sensitivity of polymerase chain reaction(PCR) technique, and direct PCR were examinded in raw milk, also were compared the calssical culture methods with PCR technique. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L nomocytogenes. In the PCR specificity tests, each of the 10 strains of L monocytogenes tested gave a single 70-bp band. But the other six Listera spp tested gave negative results. Results of the sensitivity tests showed that as few as 2 CFU of L monocytogenes in pure cultures could be detected with 16S rRNA-based primers, L-1 and L-2. In different PCR cycles, a PCR product was detected with $10^3$ cells of L monocytogenes from 25 cycles to 50 cycles and the concentration of PCR products was cycle-dependent. Raw milk samopes added L monocytogenes cells gave negative results. However, these samplers gave a single 70-bp band by pretreatment of pronase, and PCR products were detected with $10^1$ cells of L monocytogenes. To detemine the most sensitive culture protocol to use in conjunction with the PCR assay, raw milk samples were inoculated with L monocytogenes at concentrations ranging from 1 to $5.7{\times}10^4CFU/ml$. PCR assays from Listeria enrichment broth(LEB) containing raw milk samples added L monocytogene EGD could dtect 10 cells in pronase-pretreated samples without incubation, and 1 cell of L monocytogenes in both 12 hr and 24 hr incubation, respectively. Isolation raw of PCR assays was similar to that of classical culture methods, but required time for detection of L monocytogenes could remarkably be reduced compare to culture methods.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.838-848
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• 2012

Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.