• Food Science of Animal Resources
• /
• v.39 no.1
• /
• pp.13-22
• /
• 2019

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ${\beta}$-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, $pULP3-P_{LDH}$, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

• Restorative Dentistry and Endodontics
• /
• v.21 no.1
• /
• pp.403-424
• /
• 1996

Cell culture methods have been used to assess the cytotoxicity of dental materials. Different paramaters are used to monitor cytotoxic effects. But it is difficult to compare each investigator's results with different methods. The objective of this study was to investigate cytotoxic effect of several retrograde filling materials according to cell lines and assay methods. Cytotoxicity of Bestalloy (Dogmyung, Korea), Prisma APH(Densply International Inc., U.S.A.), Clearfil FII (Kuraray Co., Japan), Fuji II (GC Co., Japan), Fuji II LC (GC Co., Japan) and IRM (Densply Co., U.S.A.) on L929, 3T3 and KB permanent cell lines was measured. Radiochromium, Lactate dehydrogenase (LDH) release method and colorimetric assays, namely neutral red (NR) and MTT were used. Each material was mixed according to the manufacturer's instruction. They were tested as solid and extracted state. Cell culture media were added to each mixed or solid materials then the solution was collected and used as extract solutions. Solid Fuji II showed mild cytotoxicity on three cell lines using radiochromium release method. There was no difference in cytotoxicity of extract solution group using radiochromium release method. In colorimetric assay immediate Fuji II group and all the IRM groups showed severe cytotoxic effect. Difference in cyctotoxicity was due to rather kinds of cell lines than assay methods. Solid Fuji II and IRM showed mild cytotoxicity on three cell lines. But extract solutions had different cytotoxic effect according to cell lines using LDH release assay. Light-cured glass ionomer had mild to moderate degree of cytotoxicity on three cell lines. Cytotoxicity was affected by specimen prepaton. Susceptibility of each cell ines were also affected by assay emthods. It was suggested that cytotoxicity study using only one cell line and/or assay method might not accurately reflect the real toxic nature of dental biomaterials.

• Journal of Life Science
• /
• v.20 no.11
• /
• pp.1595-1602
• /
• 2010

Oxidation of low density lipoprotein (LDL) has been recognized as an important role in the initiation and progression of atherosclerosis. In this study, effects of allylmercaptan, a major metabolite compound of garlic, was studied on endothelial cell injury induced by oxidized low density lipoprotein (ox-LDL). The antioxidative activity of allylmercaptan was investigated by monitoring a thiobarbituric acid substance (TBARS). Allylmercaptan inhibited LDL oxidation induced by $Cu^{2+}$ at concentrations of 0.1, 1 and 10 mM in a dose dependent manner. Lactate dehydrogenase (LDH) release, as an index of cell injury, and intracellular glutathione levels were determined. Pulmonary artery endothelial cells were preincubated with allylmercaptan at $37^{\circ}C$ and 5% $CO_2$ for 24 hr, washed, and then exposed to 0.1 mg/ml oxidized LDL for 24 hr. Preincubation of endothelial cells with allylmercaptan significantly prevented the LDH release and depletion of GSH. Peroxides were measured directly in 24 well plates using a fluorometric assay. Allylmercaptan inhibited release of peroxides induced by ox-LDL in pulmonary artery endothelial cells. In a free system, allylmercaptan was shown to scavenge hydrogen peroxide. The data indicate that allylmercaptan can protect pulmonary artery endothelial cells from injury caused by oxidized LDL, and suggest that allylmercaptan may be useful for the prevention of atherosclerosis.

• Food Science and Biotechnology
• /
• v.17 no.2
• /
• pp.308-312
• /
• 2008

The flower buds of Tussilago farfara (TF) have been traditionally used in oriental medicine for the treatment of bronchitis and asthma. In our study, the primary objective was to determine the mechanisms that are inherent to TF-induced cytotoxicity and apoptosis, using the methanolic extract of TF (TFM) in HT-29 human colon cancer cells. We found that TFM-induced induced cytotoxicity in HT-29 cells in a dose-dependent manner. This effect was verified via an MTT reduction assay, an lactate dehydrogenase (LDH) release assay, and a colony formation assay. Interestingly, we also detected apoptotic bodies on Hoechst staining, and attempted to determine whether TFM-induced apoptosis involved the caspase pathway using a caspase-3/7 activity assay. Overall, the results indicate that TFM contain chemotherapeutic agents and potential candidates use for against human colon cancer cells.

• Parasites, Hosts and Diseases
• /
• v.34 no.1
• /
• pp.59-68
• /
• 1996

This study investigated the enzyme histochemical localization and characteristics of lactate (LDH) and malate dehydrogenase (MDH) related with the oxidation-reduction metabolism in the sparganum and adult of 5. erinacei. By enzyme histochemical assay, activity of LDH was strong in the tegument and subtegumental muscle layers of the adult and sparganum. Activity of MDH was strong in the tegument of the sparganum and subtegumental muscle layers of the adult. However it was weak in the tegument of the adult. By electrophoresis, 45 kDa band was major and common in LDH of adults and spargana. The 150 kDa molecule was the major and common band in MDH of adults and r -spargana (from experimentally infected rats) . By isoelectrofocusing, isoelectric points (Pl) or 4 MDH isogyme from adult worm were 6.0.6.5, 6.7 and 7.1, respectively. Pl 6.0 was the major band. The active range of pH for MDH was about pH 6-8 and the optimum pH was pH 7 The effective temperature on the MDH was about $30^{\circ}C$∼$50^{\circ}C$ and the optimum temperature was about 40℃ in spargana md adult worm. In the stability against heat, when MDH was heated at 85℃ for 10 seconds, the activity was denatured perfectly. Maximum activity or MDH was 19.4 unit in the s-sparganum (from snakes), 24.5 unit in the r-sparganum (from rats) and 108.0 unit in the adult worm. The maximum activity was higher in adults than in spargana. The present result showed us that the nutrients absorbed through the tegument were transferred into inner tissues and were utilized as the source of metabolism. According to the habitat of the parasite, the isozymes of LDH and MDH are activated differently, and by this different activation the sparganum and adult can adapt themselves to parasitic circumstances.

• Biomedical Science Letters
• /
• v.11 no.3
• /
• pp.319-326
• /
• 2005

It is well known that oxidative stress of reactive oxygen species (ROS) may be a causative factor in the pathenogenesis of bone disorder on osteoblast or osteoclast. The purpose of this study was to evaluate the cytotoxicity of oxidative stress, protective effect of glutamate receptor antagoinst against ROS-induced osteotoxicity, secretion of tumor necrosis factor $(TNF)-\alpha$ and the expression of c-fos gene in the cultured rat osteoblasts and osteoclasts. Cell viability by MTS assay or !NT assay, activity of glutathione peroxidase (GPx), lipid peroxidation (LPO) activity, protein synthesis by sulforhodamine B (SRB) assay, alkaline phosphatase (ALP) activity, lactate dehydrogenase (LDH) activity, MTS assay for NMDA (N-methyl-D-aspartate) receptor antagonist or AMPA/kainate receptor antagonist, measurement for $TNF-\alpha$, and c-fos gene expression were performed after these cells were treated with or without various cocentrations of xanthine oxidase (XO), hypoxanthine (HX), D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid (CKA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), respectively. In this study, XO/HX showed decreased cell viability and glutathione peroxidase (GPx) activity, but it showed increased LPO activity, $TNF-\alpha$ secretion and c-fos expression. APV and CKA incresed protein sythesis and ALP activity. While, CNQX or DNQX did not show any protective effect in LDH activity or cell viability. From these results, XO/HX showed cytotoxic effect in cultured rat osteoblast or osteoclast, and also NMDA receptor antagonist such as APV or CKA was effective in blocking XO/HX-induced osteotoxicity in these cultures.

• The Korean Journal of Food And Nutrition
• /
• v.28 no.6
• /
• pp.965-972
• /
• 2015

This study was conducted to evaluate the neuroprotective effects of Cheonggukjang extract in in-vitro and in-vivo models. T98G-human glioblastoma cells were pretreated with various concentrations (1~10 mg/mL) of Cheonggukjang extract for 24 h and then exposed to $H_2O_2$ (1 mM) for 3 h. The neuroprotective effects of Cheonggukjang extract were measured using a CCK-8 kit assay, total antioxidant capacity (TAC) assay, reactive oxygen species (ROS) assay, and lactate dehydrogenase (LDH) release assay. The early stage focal ischemia rodent model was used as the in-vivo neurotoxicity model. Various concentrations (10~200 mg) of Cheonggukjang extract were administered to the animal models for 1 week. Peripheral blood was analyzed for glutathione peroxidase (GPx) expression by ELISA, and infarct volume reduction was analyzed by TTC staining. Cheonggukjang extract significantly (p<0.05) increased cell viability in T98G cells against $H_2O_2$ as well as against the induced neurotoxicity. Indeed, treatment with the Cheonggukjang extract induced a decrease in ROS and LDH expression and increased TAC significantly (p<0.05). However, Cheonggukjang extract did not induce a decrease in infarct volume or an increase in GPx expression in the in-vivo model. Despite the limitation in neuroprotection, Cheonggukjang extract may be useful for treating ROS injury.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.7
• /
• pp.4267-4271
• /
• 2013

Background: As natural medicines in Asia, curcumin and triptolide extracted from different drug plants have proven to possess anticancer potential and widely used for anti-cancer research. The present study attempted to clarify that curcumin and triptolide synergistically suppress ovarian cancer cell growth in vitro. Methods: To test synergic effects, cell viability and apoptosis were analyzed after curcumin and triptolide combination treatment on ovarian cancer cell lines. Synergistic effects on apoptosis induction were determined by lactate dehydrogenase (LDH) leakage assay, intracellular reactive oxygen species (ROS) assay, mitochondrial membrane potential (MMP) loss assay and flow cytometry analysis. Critical regulators of cell proliferation and apoptosis related were analyzed by qRT-PCR and Western blotting. Results: We showed that the combination of curcumin and triptolide could synergistically inhibit ovarian cancer cell growth, and induce apoptosis, which is accompanied by HSP27 and HSP70, indicating that HSP27 and HSP70 play the important role in the synergic effect. Conclusions: From the result present here, curcumin and triptolide combination with lower concentration have a synergistic anti-tumor effect on ovarian cancer and which will have a good potential in clinical applications.

• Animal cells and systems
• /
• v.1 no.3
• /
• pp.467-473
• /
• 1997

Taurine (2-aminoethanesulfonic acid), one of bioactive amino acid in the mammalian brain, is known to exert inhibitory effects on neurons via GABA receptor. In the present study, we examined effects of taurine on glutamateinduced neurotoxicity on hippocampal neuron cell culture using cell counting method and lactate dehydrogenase (LDH) assay. After 10 d of culture, cells were stimulated with appropriate drugs. Only 43% of cultured neuronal cells survived at one day after stimulation with 500 uM L-glutamate for 10 min. Survival rate was enhanced by 82% in the presence of 10 mM taurine. LDH activity from the culture supernatant incubated with a combination of L-glutamate and taurine was less than half of that with L-glutamate alone. In the next series of experiments, interleukin-6 (IL-6) mRNA expression in cultured astrocytes was investigated using reverse tanscription-PCR (RT-PCR). IL-6 mRNA was detected in the astrocytes stimulated with L-glutamate in a dose-dependent manner, while not detected in the unstimulated control astrocytes. The expression of IL-6 mRNA caused by 10 mM glutamate was inhibited by taurine, but not by GABA. These findings demonstrated a neuroprotective action of taurine against glutamate-induced toxicity.

• Journal of Life Science
• /
• v.12 no.4
• /
• pp.490-495
• /
• 2002

Hemolysin (VFH) of V. fluvialis, which is a pathogenic bacteria, causing watery diarrhea with vomiting, abdominal croup, was purified. V. fluvialis was cultivated in BHI medium and the culture supematant was precipitated by ammonium sulfate. The protein was purified by chromatographies on columns of DEAE-cellulose and Mono-Q. Molecular weight of the purified VFH was estimated as 79kDa by SDS-PAGE. The optimal temperature for a maximum hemolytic activity was at around 35$^{\circ}C$ and the activity was decreased at 4$0^{\circ}C$ Cytotoxicity of VFH was also investigated using RTG-2 cell line. LDH assay study showed that 50$\mu\textrm{g}$/m1 of VFH release 80% of total cellular LDH (lactate dehydrogenase) from RTG-2 cell and microscopic observation also showed the morphological change of cell.