• Tuberculosis and Respiratory Diseases
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• v.43 no.1
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• pp.46-53
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• 1996

Background: Endotoxin or lipopolysaccharide(LPS) can prime phagocytic cells such as polymorphonuclear leukocytes, monocytes or animal peritoneal macrophages to generate increased amounts of secretory products such as oxygen free radicals and tumor necrosis factor, which play an important role in developing adult respiratory distress syndrome in gram negative sepsis. Human alveolar macrophages(HAM) are continuously exposed to various stimuli inhaled into the alveoli, and the response to LPS might be different in HAM. Therefore, we investigated the effect of LPS pre-exposure on HAM adhered to plastic surface and A549 cell(type II human alveolar epithelial cell line) monolayer. Methods: HAM were isolated from bronchoalveolar lavage fluid from normal lung of the patients with localized lung cancer and esophageal cancer. LPS was exposed to HAM for 2hrs before or after adherence to plastic surface of 24-well Linbro plate and A549 cell monolayer. And then HAM was stimulated with PMA(phorbol myristate acetate) or fMLP(N-formyl-methionylleucyl-phenylalanine). The amount of hydrogen peroxide($H_2O_2$) production in the supernatant was measured on the principle of peroxidase-dependent oxidation of phenol red by hydrogen peroxide. Results: LPS pre-exposure could not enhance $H_2O_2$ production in neither HAM adhered to plastic surface nor one to A549 cell monolayer. But LPS even in the absence of PMA or fMLP stimulation directly increased $H_2O_2$ release in HAM if added after the adherence to A549 cell monolayer. Conclusion: Endotoxin does not prime HAM, but may directly activate HAM adhered to alveolar epithelial cells. Further investagation will be necessary.

• Tuberculosis and Respiratory Diseases
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• v.79 no.3
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• pp.143-152
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• 2016

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the accumulation of excessive fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor ${\beta}1$ (TGF-${\beta}1$)-induced epithelial-to-mesenchymal transition (EMT) is thought to be a possible source of fibroblasts/myofibroblasts in IPF lungs. We have previously reported that apolipoprotein A1 (ApoA1) has anti-fibrotic activity in experimental lung fibrosis. In this study, we determine whether ApoA1 modulates TGF-${\beta}1$-induced EMT in experimental lung fibrosis and clarify its mechanism of action. Methods: The A549 alveolar epithelial cell line was treated with TGF-${\beta}1$ with or without ApoA1. Morphological changes and expression of EMT-related markers, including E-cadherin, N-cadherin, and ${\alpha}$-smooth muscle actin were evaluated. Expressions of Smad and non-Smad mediators and TGF-${\beta}1$ receptor type 1 ($T{\beta}RI$) and type 2 ($T{\beta}RII$) were measured. The silica-induced lung fibrosis model was established using ApoA1 overexpressing transgenic mice. Results: TGF-${\beta}1$-treated A549 cells were changed to the mesenchymal morphology with less E-cadherin and more N-cadherin expression. The addition of ApoA1 inhibited the TGF-${\beta}1$-induced change of the EMT phenotype. ApoA1 inhibited the TGF-${\beta}1$-induced increase in the phosphorylation of Smad2 and 3 as well as that of ERK and p38 mitogen-activated protein kinase mediators. In addition, ApoA1 reduced the TGF-${\beta}1$-induced increase in $T{\beta}RI$ and $T{\beta}RII$ expression. In a mouse model of silica-induced lung fibrosis, ApoA1 overexpression reduced the silica-mediated effects, which were increased N-cadherin and decreased E-cadherin expression in the alveolar epithelium. Conclusion: Our data demonstrate that ApoA1 inhibits TGF-${\beta}1$-induced EMT in experimental lung fibrosis.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.6 no.1
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• Journal of Life Science
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• v.30 no.8
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• pp.701-707
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• 2020

Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and could be an effective way of shortening the time required.

• Natural Product Sciences
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• v.8 no.1
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• pp.1-15
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• 2002

An International Cooperative Biodiversity Group (ICBG) program based at the University of Illinois at Chicago initiated its activities in 1998, with the following specific objectives: (a) inventory and conservation of of plants of Cuc Phuong National Park in Vietnam and of medicinal plants of Laos; (b) drug discovery (and development) based on plants of Vietnam and Laos; and (c) economic development of communities participating in the ICBG project both in Vietnam and Laos. Member-institutions and an industrial partner of this ICBG are bound by a Memorandum of Agreement that recognizes property and intellectual property rights, prior informed consent for access to genetic resources and to indigenous knowledge, the sharing of benefits that may arise from the drug discovery effort, and the provision of short-term and long-term benefits to host country institutions and communities. The drug discovery effort is targeted to the search for agents for therapies against malaria (antimalarial assay of plant extracts, using Plasmodium falciparum clones), AIDS (anti-HIV-l activity using HOG.R5 reporter cell line (through transactivation of the green fluorescent protein/GFP gene), cancer (screening of plant extracts in 6 human tumor cell lines - KB, Col-2, LU-l, LNCaP, HUVEC, hTert-RPEl), tuberculosis (screening of extracts in the microplate Alamar Blue assay against Mycobacterium tuberculosis $H_{37}Ra\;and\;H_{37}Rv),$ all performed at UIC, and CNS-related diseases (with special focus on Alzheimer's disease, pain and rheumatoid arthritis, and asthma), peformed at Glaxo Smith Kline (UK). Source plants were selected based on two approaches: biodiversity-based (plants of Cuc Phuong National Park) and ethnobotany-based (medicinal plants of Cuc Phuong National Park in Vietnam and medicinal plants of Laos). At mc, as of July, 2001, active leads had been identified in the anti-HIV, anticancer, antimalarial, and anti- TB assay, after the screening of more than 800 extracts. At least 25 biologically active compounds have been isolated, 13 of which are new with anti-HIV activity, and 3 also new with antimalarial activity. At GSK of 21 plant samples with a history of use to treat CNS-related diseases tested to date, a number showed activity against one or more of the CNS assay targets used, but no new compounds have been isolated. The results of the drug discovery effort to date indicate that tropical plant diversity of Vietnam and Laos unquestionably harbors biologically active chemical entities, which, through further research, may eventually yield candidates for drug development. Although the substantial monetary benefit of the drug discovery process (royalties) is a long way off, the UIC ICBG program provides direct and real-term benefits to host country institutions and communities.

• Journal of Biosystems Engineering
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• v.6 no.1
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• pp.39-47
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• 1981

For purpose of investigation the proper paddy field condition in performance of rice transplanter according to the various elapsed times (0.5, 1, 1.5, 2 days) after puddling and plowing (12, 15, 18 cm depth), this experiment was carried out on the paddy field located in Chil Am Dong, Jin Ju City, from April to May in 1980. The results are summarized as follows; 1. The practical working power for the elapsed time 0.5 days and plowing depth 18cm was about 0.8 ps, which was the highest among the plots, so the power out-put(2.5~3.5 p.s) of these engines are considered to be enough for the transplanting under these field conditions. 2. The percentage of slip increased proportional1y to the plowing depth and decreased proportionally to the elapsed time after puddling, and the highest and lowest percentages of slip were 42.5% in elapsed time 0.5 days, plowing depth 18 cm, and 26.5% in elapsed time 2 days, plowing depth 12 cm, respectively. 3. In the plot of elapsed time 2 days and plowing depth 12 cm, the planting distance was 13.9 cm, which was closed to the proper planting distance 14 cm. 4. The percentage of missing hill was lowest(1.5%) in the plot of elapsed time 2 days and plowing depth 12 cm. 5. The planted depth in the plot of the elapsed time 2 days and plowing depth 15 cm was 2. 95 cm, which was closed to the proper planting depth 3 cm. 6. The angle of planting postures in the plot of elapsed time 2 days and plowing depth 12 cm was $89^{\circ}$, which was closed to the desirable posture angle $90^{\circ}$. 7. The deviation from the straight transplanting line was lowest in the plot of the elapsed time 2 days and plowing depth 12 cm. 8. From the results above mentioned, it is recommended that the field condition under the elapsed time 2 days and plowing depth 12 cm is the most favorable one for the working performance of rice thansplanter.

• The Korean Journal of Pharmacology
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• v.21 no.2
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• pp.128-141
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• 1985

To investigate the influence of phenobarbital sodium on the action of morphine and on the diurnal rhythms of both opiate receptor binding and ${\beta}-endorphin$ contents, the amount of specifically bound $(^3H)$-morphine and immunoreactive ${\beta}-endorphin$ were measured in the midbrain of phenobarbital-treated rats at 4h intervals in a day. Rats were housed and adapted to a controlled cycle of either 12 h light-12 h dark or 24 h constant dark. After 3 weeks of adaptation, 0.5 ml of physiological saline or phenobarbital sodium (20mg/kg/day, i.p.) were administered twice a day for 2 weeks. Highly significant diurnal rhythms of opiate receptor binding and ${\beta}-endorphin$ were present in rat midbrain. In control group, the peak of maximum $(^3H)$-morphine binding was observed at 22:00 h, whereas the peak of ${\beta}-endorphin$ content was found at 06:00 h. Even in the absence of time cues these diurnal rhythms persisted, but they were highly modified with respect to the wave form as well as differences in the timing of peak and nadir. In the phenobarbital-treated group, these diurnal rhythms were also modified in shape, phase and amplitude, as well as in timing of peak and nadir. In this group, 24 h mean of opiate receptor binding was significantly decreased, while the 24 h mean level of ${\beta}-endorphin$ content was highly increased. However, Kd values in all experimental groups did not change. This indicates that differences in binding were not due to changes in the affinity, but in the number of binding sites. Statistical analysis of regression line indicates that changes of receptor binding were closely correlated with the changes of ${\beta}-endorphin$ content. These results suggest that phenobarbital may influence the action of morphine by changing the number of opiate receptors and that the modification of diurnal rhythm of opiate receptor by the agent is possibly due to changes of ${\beta}-endorphin$ content.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.5 no.1
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• pp.59-69
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• 2000

Environmental factors and phytoplankton community have been bimonthly investigated in order to clarify the bioecological characteristics of coral habitats around Moonsom at the southern Cheju Island from September 1995 to July 1996. Annual mean temperature and annual mean salinity were $17.4^{\circ}C$ and 34.06 psu, respectively, showing lower temperature-higher salinity in winter and higher temperature-lower salinity in summer, which means such conditions are inadequate for coral reef formation. Nutrient concentrations represent that total nitrogen ranged from $0.07{\sim}10.08\;{\mu}M$, phosphate from $0.05{\sim}1.70\;{\mu}M$, and silicate from $3.08{\sim}21.86\;{\mu}M$. The N/P ratio showed the range of 9.59-10.60 with decreasing offshore-ward, which means the phytoplankton community could be limited by nitrogen sources. Annual mean euphotic depth was 32.0m (18.9m-48.6m) with difference according to season and reveals the close relationship with the depth of coral distribution. Chlorophyll a concentrations of phytoplankton ranged from $0.12{\sim}1.51\;{\mu}g\;L^{-1}$ and standing crops from $1.5{\times}10^3{\sim}7.0{\times}10^5\;cells\;L^{-1}$, showing higher at inshore than at offshore with a blooming in May. A total of 128 species of phytoplankton occurred in all stations, representing 99 spp. of diatoms, 26 spp. of dinoflagellates, 2 spp. of silicoflagellates and 1 sp. of blue-green algae. Diatoms are main taxa in all seasons except for occupying by dinoflagellates in summer. Among dominant species, fParalia sulcata (Ehrenberg) Cleve and Cylindrotheca closterium (Ehrenberg) Lewin & Reimann were predominant and are likely to be main food sources for coral community. Annual mean species diversity index (H') was 1.84, showing lower than around the coast line of Cheju Island.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.344-360
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• 2017

Zoysiagrasses are important turf plants used for school playgrounds, parks, golf courses, and sports fields. The two most popular zoysiagrass species are Zoysia japonica and Zoysia sinica. These are widely distributed across different growing zones and are morphologically distinguishable from each other; however, it is phenotypically difficult to differentiate those that grow along the coastal line from those in beach area habitats. A combination of morphological and molecular approaches is desirable to efficiently identify these two plant cultivars. In this study, we used a rapid identification system based on DNA barcoding of the nrDNA-internal transcribed spacer (ITS) regions. The nrDNA-ITS regions of ITS1, 5.8S nrDNA, and ITS2 from Z. japonica, Z. sinica, Agrostis stolonifera, and Poa pratensis were DNA barcoded to classify these grasses according to their molecular identities. The nrDNA-ITS sequences of these species were found at 686 bp, 687 bp, 683 bp, and 681 bp, respectively. The size of ITS1 ranged from 248 to 249 bp, while ITS2 ranged from 270 to 274 bp. The 5.8S coding region ranged from 163 - 164bp. Between Z. japonica and Z. sinica, nineteen (2.8%) nucleotide sites were variable, and the G+C content of the ITS region ranged from 55.4 to 63.3%. Substitutions and insert/deletion (indel) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted to cleaved amplified polymorphic sequence (CAPS) markers, and applied to the Zoysia grasses sampled to verify the presence of these markers. Among the 62 control and collected grass samples, we classified three groups: 36 Z. japonica, 22 Z. sinica, and 4 Z. japonica/Z. sinica hybrids. Morphological classification revealed only two groups; Z. japonica and Z. sinica. Our results suggest that used of the nrDNA-ITS barcode region and CAPS markers can be used to distinguish between Z. japonica and Z. sinica at the species level.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.