• The Korean Journal of Mycology
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• v.16 no.2
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• pp.64-69
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• 1988

Isoates with changed pathogenicity were selected among iprodione-resistant Alternaria mali to investigate any relationship between their pectolytie enzyme activity and pathogenicity. In artificial medium, strongly pathogenic isolates $S_1$, and $R_3$ showed higher enzyme activity than weakly pathogenic isolate $R_8$.Activity of endo-polymethylgalacturonase and end-o-polygalacturonase was more than 3 times. But activity of pectin methylesterase and pectin Iyase by isolaste $S_1$ was higher than those by $R_3$ and $R_8$ isolates. In apple medium dialyzed against distilled water, activity of enzyme by each isolate was increased but growth of each isolate was reduced. When iprodione was added to the medium, enzyme activity and growth of isolate $S_1$, were reduced but strongly pathogenic isolate .$R_3$ among iprodione-resistant ones showed increased enzyme activity except for exo-polygalacturonase in dialyzed apple medium.

• The Korean Journal of Mycology
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• v.18 no.1
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• pp.7-12
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• 1990

Isolates of Penicillium expansum with reduced pathogenicity were arbitrarily selected among benomyl-resistant isolates in order to investigate relationship of their pectolytic enzyme acitivity with pathogenicity. In artificial medium, strongly pathogenic isolate $S_1$ and weakly pathogenic isolate $R_2$ produced considerable amonts of endo-polymethylgalacturonase, endo-polygalacturonase, pectin methyl-trans-eliminase, and polygalacturonate-trans-eliminase. No marked difference in enzyme activities was observed between two isolates. In apple medium, the activities of endo-polymethylgalacturonase and endo-polygalacturonase of isolate $S_1$ were over 6 times higher than those of isolate $R_2$. But pectin methyl-trans-eliminase and polygalacturonate-trans-­eliminase did not show a great difference. Activities of endo-polymethylgalacturonase and endo­polygalacturonase precipitated at 80-95% saturation of ammonium sulfate were highest, and addition of these enzyme solutions increased pathogenicity of weakly pathogenic isolates $R_{1-4}$.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.6
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• pp.434-439
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• 2002

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. OSCC generally has a poor prognosis due to its tendency towards a local invasion and subsequent metastasis, which is mediated by multiple proteolytic enzymes and angiogenesis. Soy products contain high levels of isoflavonoids, including the tyrosine kinase inhibitor, genistein, which has been identified as a potent inhibitor of cell proliferation and in vitro angiogenesis. The purpose of this in vitro study is to evaluate the anti-cancer effect of genistein with respect to the angiogenesis and basement membrane invasion in OSCC. The highly invasive OSCC cell line, HSC-3 cells were cultured in the presence of $10{\mu}M$ genistein for 24h. To evaluate the effects of genistein on the invasiveness and the gelatinolytic activity, in vitro invasion assay and zymography were performed. In order to evaluate the effect on the VEGF and bFGF mRNA expression, RT-PCR and northern hybridization reaction, and chemiluminescence detection were applied. The in vitro invasion assay showed that the genistein treatment reduced the cellular invasion through the artificial basement membrane and significant difference between the control group and the genistein treated group was shown in MMP-2 activity. Especially, the 62 kDa activated form of MMP-2 in the control group was 1.8 times higher than that in the genistein treated group. The results of the northern blot analyses indicated that VEGF mRNA expression in the genistein treated group was significantly down regulated. This study showed that genistein inhibits angiogenesis and reduces basement membrane invasion in OSCC. It seems to support the possibility of genistein as an anti-cancer agent.

• Journal of Sericultural and Entomological Science
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• v.41 no.2
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• pp.108-115
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• 1999

An improvement of methods in fibroin hydrolysates preparat significantly enlarges their applications to practical use. Acidic hydrolysis by hydrohloric acid is one of the methods for silk fibroin depolymeriza6tion. A low yield of final product and long time of the process are the demerits of this method. Possibility of preparation of water-soluble silk hydrolysates with more yield and less expenses is investigated in this study. Such possibility is occurred with the increasing tratment temperature and simultaneously decreasing treatment time, concentration of hydrochoric acid respectively, the concentration of sodium hydroxide used for neutraliza6tion of hydolysates after hydrolysis. Colour is decreased in this case and a small amount of activated, too. Protection of hydrolysates against precipitation after neutraliza6tion, and separation and during concentrating process is the other merit of this method. Creamy-coloured insoluble silk powder is the remainder of hydrolyzed fibroin. This is the only the immobiliza6tion of enzymes and other physiological active substances. Fine particles of this powder can be used as additives for artificial diets and cultural media, as well as raw materials for polymer membranes, etc.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.501-508
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• 2003

Artificial coupling of one enzyme with another can provide an efficient means for the production of industrially important chemicals. Xylose reductase has been recently discovered to be useful in the reductive production of xylitol. However, a limitation of its in vitro or in vivo use is the regeneration of the cofactor NAD(P)H in the enzyme activity. In the present study, an efficient process for the production of xylitol from D-xylose was established by coupling two enzymes. A NADH-dependent xylose reductase (XR) from Pichia stipitis catalyzed the reduction of xylose with a stoichiometric consumption of NADH, and the resulting cofactor $NAD^+$ was continuously re-reduced by formate dehydrogenase (FDH) for regeneration. Using simple kinetic analyses as tools for process optimization, suitable conditions for the performance and yield of the coupled reaction were established. The optimal reaction temperature and pH were determined to be about $30^{\circ}C$ and 7.0, respectively. Formate, as a substrate of FDH, affected the yield and cofactor regeneration, and was, therefore, adjusted to a concentration of 20 mM. When the total activity of FDH was about 1.8-fold higher than that of XR, the performance was better than that by any other activity ratios. As expected, there were no distinct differences in the conversion yields of reactions, when supplied with the oxidized form $NAD^+$ instead of the reduced form NADH, as a starting cofactor for regeneration. Under these conditions, a complete conversion (>99%) could be readily obtained from a small-scale batch reaction.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.261-271
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• 2019

Background: Protopanaxatriol (PPT) is an aglycone of ginsenosides, which has high medicinal values. Production of PPT from natural ginseng plants requires artificial deglycosylation procedures of ginsenosides via enzymatic or physicochemical treatments. Metabolic engineering could be an efficient technology for production of ginsenoside sapogenin. For PPT biosynthesis in Panax ginseng, damarenediol-II synthase (PgDDS) and two cytochrome P450 enzymes (CYP716A47 and CYP716A53v2) are essentially required. Methods: Transgenic tobacco co-overexpressing P. ginseng PgDDS, CYP716A47, and CYP716A53v2 was constructed via Agrobacterium-mediated transformation. Results: Expression of the three introduced genes in transgenic tobacco lines was confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). Analysis of liquid chromatography showed three new peaks, dammarenediol-II (DD), protopanaxadiol (PPD), and PPT, in leaves of transgenic tobacco. Transgenic tobacco (line 6) contained $2.8{\mu}g/g$ dry weight (DW), $7.3{\mu}g/g$ DW, and $11.6{\mu}g/g$ DW of PPT, PPD, and DD in leaves, respectively. Production of PPT was achieved via cell suspension culture and was highly affected by auxin treatment. The content of PPT in cell suspension was increased 37.25-fold compared with that of leaves of the transgenic tobacco. Transgenic tobacco was not able to set seeds because of microspore degeneration in anthers. Transmission electron microscopy analysis revealed that cells of phloem tissue situated in the center of the anther showed an abnormally condensed nuclei and degenerated mitochondria. Conclusion: We successfully achieved the production of PPT in transgenic tobacco. The possible factors deriving male sterility in transgenic tobacco are discussed.

• BMB Reports
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• v.56 no.4
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• pp.258-264
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• 2023

As a high-grade soft-tissue sarcoma (STS), undifferentiated pleomorphic sarcoma (UPS) is highly recurrent and malignant. UPS is categorized as a tumor of uncertain differentiation and has few options for treatment due to its lack of targetable genetic alterations. There are also few cell lines that provide a representative model for UPS, leading to a dearth of experimental research. Here, we established and characterized new cell lines derived from two recurrent UPS tissues. Cells were obtained from UPS tissues by mincing, followed by extraction or dissociation using enzymes and culture in a standard culture environment. Cells were maintained for several months without artificial treatment, and some cell clones were found to be tumorigenic in an immunodeficient mouse model. Interestingly, some cells formed tumors in vivo when injected after aggregation in a non-adherent culture system for 24 h. The tissues from in vivo study and tissues from patients shared common histological characteristics. Pathways related to the cell cycle, such as DNA replication, were enriched in both cell clones. Pathways related to cell-cell adhesion and cell-cell signaling were also enriched, suggesting a role of the mesenchymal-to-epithelial transition for tumorigenicity in vivo. These new UPS cell lines may facilitate research to identify therapeutic strategies for UPS.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.16-16
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• 2015

Ectomycorrhizal (ECM) mushrooms play a major role in plant growth promotion through symbiotic association with roots of forest trees. They also provide an economically important food resource to us and therefore they have been studied for their artificial cultivation for decades in Korea. We have secured bio-resources of ECM mushrooms from Korean forests and performed their physiological studies. To investigate the cultural characteristics, the fungi were cultured under different conditions (medium, temperature, pH of the medium, inorganic nitrogen source). More than 90% of total 160 strains grew on three solid media (potato dextrose agar, PDA; sabouraud dextrose agar, SDA; modified Melin-Norkrans medium, MMN). The rate of mycelial growth on malt extract agar (MEA) was lower than those of three media (PDA, SDA, MMN). None of the Tricholomataceae strains grew on MEA. Many strains of ECM mushrooms were able to grow at the temperature range of $15{\sim}25^{\circ}C$ on PDA, while they showed poor growth at $10^{\circ}C$ or $30^{\circ}C$. In particular, the growth rates of both Gomphaceae and Tricholomataceae were significantly lower at $10^{\circ}C$ than at $30^{\circ}C$. The optimal pH of many strains was pH 5.0 when they cultured in potato dextrose broth (PDB). Fifty-seven percent of tested strains grew well on medium containing ammonium source than nitrate source. Many strains of Tricholomataceae showed a notable growth on ammonium medium than nitrate medium. Twenty-three percent of strains preferred nitrate source than ammonium source for their mycelial growth. The production and activity of two enzymes (cellulase and laccase) by ECM fungi were also assayed on the enzyme screening media containing CMC or ABTS. Each strains exhibited different levels of enzymatic activities as well as enzyme production. The number of laccase-producing strains was less than that of cellulase-producing strains. We found that 77% of tested strains produced both cellulase and laccase, whereas 2% of strains did not produce any enzymes. The morphological characteristics of mycelial colony were also examined on four different solid media. Yellow was a dominant color in mycelial colony and followed by white and brown on all culture media. ECM mushrooms formed mycelial colonies with a single or multiple colors within a culture medium depending on the strains and culture media. The most common shape of mycelial colony was a circular form on all media tested. Other families except for Amanitaceae formed an irregular colony on MMN than PDA. All strains of Tricholomataceae did not form a filamentous colony on all media. The pigmentation of culture media by mycelial colonies was observed in more than 50% of strains tested on both PDA and SDA. The degree of pigmentation on PDA or SDA was higher than MMN and brown color was dominant than yellow color. The production of exudates from mycelial colony was higher on PDA than MMN. Brown exudates were mainly produced by many strains on PDA or SDA, whereas transparent exudates were mainly produced by strains on MMN. We observed the mycelial colonies with a single or multiple textures in just one culture plate. Wrinkled or uneven colony surfaces were remarkably observed in many strains on PDA or SDA, while an even colony surface was observed in many strains on MMN. Sixty percent of Tricholomaceae strains formed wrinkled surface on PDA. However, they did not form any wrinkle on MMN plate. Cottony texture was observed in mycelia colonies of many strains. Velvety texture was often observed in the mycelial colonies on SDA than PDA and accounted for 60% of Suillaceae strains on SDA.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

• Food Science and Preservation
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• v.11 no.3
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• pp.417-423
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• 2004

Botanical antimicrobial agent-grapefruit seed extract mixture (BAAG) have an unknown compounds which exhibit the antibiotic activities aganist microorganisms including bacteria and fungi. We have examined the effects of BAAG on the physiological function of Botrytis cinerea which was isolated from necrotic lesions of decayed fruits and vegetables such as cucumbers, grapes, tomatoes, and red peppers during storage. In the results of enzymatic activities related to the energetic metabolism there was no inhibitory effect of BAAG on the activities of several enzymes in vitro including glucose 6-phosphate dehydrogenase and malate dehydrogenase, while there was inhibitory effect of BAAG on the activities of hexokinase and succinate dehydrogenase. O-nitrophenyl-$\beta$-D-galactopyranoside(ONPG), the artificial substrate of $\beta$-galactosidase was hydrolyzed in the presence of BAAG, indicating that the membrane was pertubated by the BAAG. From the results we suggested that the antibiotic activity of BAAG is due to the change of membrane permeability of the cell. BAAG was fractionated and purified by silica gel and sephadex column chromatography. Among active fractions two peaks were identified as naringin and limonin when they were analyzed by by NMR and Fast atomic bombardment.