• Molecules and Cells
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• v.37 no.9
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• pp.656-663
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• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.

• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• Applied Biological Chemistry
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• v.13 no.1
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• pp.43-50
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• 1970

To study the productivity of single cell protein from the petroleum hydrocarbon utilizing yeasts, 242 soil samples, such as oil soaked soil of gas stations and garage, coal, farm soil, and sewage, from 135 places in Korea were collected. From these samples 468 yeast strains which utilize petroleum hydrocarbon as a sole organic carbon source were isolated and identified by observing the growth rates. For the identified strains optimum culture conditions were determined and analysis of cell components were performed. 1. 90.8% of petroleum hydrocarbon utilizing yeast strains were found from oil soaked soil and about 10% from coal, farm soil and sewage etc. 2. The yeast strain of the highest cell productivity was isolated from oil soaked soil and was identified as Candida curvata HY-69-19. 3. The optimum culture conditions for the selected yeast strain were found to be pH 5.0, $28^{\circ}C$ and affluent aerated state. 4. Candida curvata HY-69-19 was found to utilize favorably the heavy gas oil fractionated at above $268.9^{\circ}C$ as carbon source and urea as inorganic nitrogen source. 5. The growth curve of this strain on heavy gas oil medium showed that the yeast has a lag phase up to 18 hours and logarithmic growth phase between 24 to 42 hours. Generation time was found to be between 3.8 and 4.5 hours during the logarithmic growth phase. 6. About 300 mg dried cells per heavy gas oil was harvested under the culture conditions of adjusted pH to 5.0 at time intervals of 6 hours for 54 hours and heavy gas oil urea for shaking culture medium. 7. Chemical composition of the yeast cell was found to be 40.25%, 14.81%, 24.32% and 10.63% for crude protein, crude lipid, carbohydrate and ashes, respectively.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.488-495
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• 2000

Strain SA72 was isolated from Jeot-gal and identified as producer of a bacteriocin, which showed some bactericidal activity against Lactobacillus delbrueckii ATCC 4797. Strain SA72 was tentatively identified as Lactococcus lactis according to the AOI test. Lactococcus lactis SA72 showed a broad spectrum of microorganisms, tested by the modified deferred method. The activity of lacticion SA72, named tentatively as a bacteriocin produced by Lactococcus lactis SA72, was detected during the mid-lon growth phase, reached a maximum during the early stationary phase, and then declined after the late stationary phase. Lacticin SA72 also showed a relatively broad spectrum of activity against non-pathogenic and pathogenic microorganisms when assessed by the spot-on-lawn method. Its anitimicrobial activity on sensitive indicator cells disappeared completely by protease XIV treatment. The inhibitory activity of lacticin SA72 remained after treatment for 15 min at $121^{\circ}C$, 문 was stable in a pH range of 2.0 to 9.0 and all organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against Lactobacillus delbrueckii ATCC 4797. The apparent molecular mass of lacticin SA72 was in the region of 3-3.5 kDa, determined by SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.231-236
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• 1992

A 5.8 kb chromosomal DNA fragment of Serratia marcescens ATCC 27117 including an extracellular serine protease gene was cloned in Escherichia coli. The cloned gene(pSMP18) caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells. The protease purified from E. coli harboring pSMP18 was inactivated not by 100 mM EDTA but by 10 mM phenyl methyl sulfonyl flouride (PMSF). The molecular weight of the purified serine protease was about 66, 000 in the SDS-PAGE and the isoelectric point was approximately 5.7 in IEFㆍGel electrophoresis. The optimal pH and temperature for reaction of the purified serine protease were 9.5 and $45^\circ{C}$, respectively.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.4
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• pp.241-244
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• 2011

A lectin from the hemolymph of purse crab, Philyra pisum, was found to have anti-proliferative activity on human lung cancer cells by our laboratory. In this study, P. pisum lectin (PPL) was molecularly characterized including molecular mass, amino acid sequences, amino acid composition, and the effects of metal ions, temperature, and pH on the activity. We found that PPL showed mitogenic activity on human lymphocytes and BALB/c mouse splenocytes. The mitogenic activity (maximum stimulation index, $SI=9.57{\pm}0.59$) of PPL on human lymphocytes was higher than that of a standard well-known plant mitogen, concanavalin A (maximum $SI=8.80{\pm}0.59$). The mitogenic activity mediated by PPL is required for optimum dosing, and higher or lower concentrations caused decreases in mitogenic response. PPL also induced mitogenic activity on mouse splenocytes, however, the maximum SI ($1.77{\pm}0.09$) on mouse splenocytes of PPL was lower than that ($2.14{\pm}0.15$) of concanavalin A. In conclusion, PPL is a metal ion-dependent monomer lectin with mitogenic activity, and could be used as a lymphocyte or splenocyte stimulator.

• Journal of Life Science
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• v.26 no.3
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• pp.289-295
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• 2016

Sorbus commixta, a flowering plant in the Rosaceae family, is native to Japan and Ulleung Island, Korea. This plant is also called maga-mok or mai-mok in Korea because the bud of the stem has a similar shape to the teeth of a horse. In this study, hot water extracts from different parts of S. commixta, such as leaf, stem, and immature and mature fruits, were prepared, and their antithrombosis and antioxidant activities were evaluated. The extraction yield and pH of stem extracts were 3.99% and 5.5, respectively. The stem extracts contained 89.2 mg/g of total polyphenols and 28.3 mg/g of total flavonoids. The hot water extracts prepared from the leaf, stem, immature, and mature fruit of S. commixta exhibited no hemolytic activity against human red blood cells, up to a concentration of 0.5 mg/ml. In an anticoagulation assay, the stem extracts showed strong extension in thrombin, prothrombin, and activated partial thromboplastin times, whereas the other extracts had no anticoagulation activity. In a platelet aggregation inhibitory activity assay, all the extracts tested had no inhibitory activity against human platelets. With regard to antioxidation activity, the stem extracts showed stronger radical scavenging activity and reducing power activity than the other extracts. The calculated RC₅₀s, the concentration required for 50% radical scavenging activity, for DPPH anions, ABTS cations, and nitrite of the crude stem extracts were 119.7, 53.3, and 117.5 μg/ml, respectively, whereas they were 13.7, 5.2, and 14.9 μg/ml for DPPH anions, ABTS cations, and nitrite, respectively, for vitamin C. The results suggest that the stem extracts of S. commixta have strong potential for use as a novel resource for antithrombosis agents.

• Journal of Oral Medicine and Pain
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• v.36 no.3
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• pp.147-160
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• 2011

Eugenol (4-allyl-2-methoxyphenol) is a natural phenolic constituent extensively used in dentistry as a component of zinc oxide eugenol cement and is applied to the mouth environment. Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and natural phenolic compound, eugenol on SCC25 human tongue squamous cell carcinoma cell line. To investigate whether the co-treatment with eugenol and CGM compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by Hoechst staining, TUNEL staining and DNA hypoploidy. Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and the translocation of apoptosis-related proteins in co-treatment. In this study, co-treatment of with eugenol and CGM on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, the increase and decrease of Bax and Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-3, caspase-6 caspase-7, caspase-9, PARP, Lamin A/C and DFF45 (ICAD) whereas each single treated SCC25 cells did not show or very slightly these patterns. Although the single treatment of 40 ${\mu}g$/ml CGM and 0.5 mM eugenol for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that combination therapy with CGM and eugenol could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.

• Journal of fish pathology
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• v.4 no.1
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• pp.31-39
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• 1991

Vibrio sp. S-1-2 was isolated from seawater in the Masan bay and its bacterial characteristics and bioaccumulation of $CdCl_2$ in the cell were investigated. As the result of microscopic and biochemical test, the S-1-2 strain was identified to Vibrio sp. and this strain can be tolerated even in 200 ppm $CdCl_2$ media, however its growth was inhibited. In 25 ppm $ZnCl_2$ media, the growth of Cd resistant Vibrio sp. S-1-2 was promoted at later stage of growth. The growth of Vibrio sp. S-1-2 was inhibited on 25 ppm $CuCl_2$ and $PbCl_2$ media and was not able to grow in 25 ppm $HgCl_2$ media at all. The uptake of cadium in the cells was increased exponentially with increasing concentration of $CdCl_2$ in media. But the uptake rate of cadmium was suddenly inhibited at 50 ppm $CdCl_2$ media. Optimal pH and NaCl concentration for bioaccumulation of $CdCl_2$ were 8-9 and 1-2%, respectively. In the case of pH, maximum dpm value was found at pH 7 after 96 hours culture and in the case of NaCl concentration, it was detected in 2% NaCl media after 36 hours culture.