• Restorative Dentistry and Endodontics
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• v.30 no.5
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• pp.409-422
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• 2005

The aim of this study was to identify the bacteria isolated from acute endodontic lesions by cell culture and 16S rDNA sequencing. The necrotic pulpal tissue was collected from 17 infected root canals, which were diagnosed as being either an acute pulpitis or acute periapical abscess. Samples were collected aseptically from the infected pulpal tissue of the infected root canals using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ul of 1 XPBS. The sample solution was briefly mixed and plated onto a BHI-agar plate containing $5\%$ sheep blood. The agar plates were incubated in a $37^{\circ}C$ anaerobic chamber for 7 days. The bacteria growing on the agar plate were identified by 16S rRNA coding gene (rDNA) cloning and sequencing at the species level. Among the 71 colonies grown on the agar plates, 56 strains survived and were identified. In dental caries involving the root canals, Streptococcus spp. were mainly isolated. Actinomyces, Clostridia, Bacteroides and Fusobacteria were isolated in the periapical lesion without dental caries. Interestingly, two new Actinomyces spp. (ChDC B639 and ChDC B631) were isolated in this study. These results showed that there was diversity among the species in endodontic lesions, This suggests that an endodontic infection is a mixed infection with a polymicrobial etiology. These results may offer the bacterial strains for pathogenesis studies related to an endodontic infection.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.13-13
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• 2018

The anti-inflammatory (INF) compounds (1-15) were isolated from Agrimonia pilosa Ledeb. (APL) by activity-guided isolation technique. The isolated compounds (1-15) were identified as quercetin-7-O-rhanmoside (1), apigenin-7-O-glycoside (2), kaempferol-7-O-glycoside (3), apigenin-7-O-[6"-(butyl)-glycoside] (4), querceitn (5), kaempferol (6), apigenin (7), apigenin-7-O-[6"-(pentyl)-glycoside] (8), agrimonolide (9), agrimonolide-6-O-glucoside (10), desmethylagrimonolide (11), desmethylagrimonolide-6-O-glucoside (12), luteolin (13), vitexin (14) and isovitexin (15). Flavonoids, compound 2, 3, 11, and 14-15 have been found in APL for the first time. Furthermore, two novel flavone derivatives, compound 4 and 8, have been isolated inceptively in plant. In the no cytotoxicity concentration ranges of $0-20{\mu}M$, nitric oxide (NO) production level of 1-15 was estimated in LPS-treated Raw 264.7 macrophage cells. The flavone aglycones, 7 (apigenin, $IC_{50}=3.69{\pm}0.34{\mu}M$), 13 (luteolin, $IC_{50}=4.62{\pm}0.43{\mu}M$), 6 (kaempferol, $IC_{50}=14.43{\pm}0.23{\mu}M$) and 5 (quercetin, $IC_{50}=19.50{\pm}1.71{\mu}M$), exhibited excellent NO inhibitory (NOI) activity in dose-dependent manner. In the structure activity relationship (SAR) study of apigenin-derivatives (APD), apigenin; Api, apigenin-7-O-glucoside; Api-G, apignenin-7-O-[6"-(butyl)-glycoside]; Api-BG and apignenin-7-O-[6"-(pentyl)-glycoside]; Api-P, from APL on INF activity was investigated. The INF mediators level such as NO, INF-cytokines, NF-KB proteins, iNOS and COX-2 were sharply increased in Raw 264.7 cells by LPS. When pretreatment with APD in INF induced macrophages, NOI activity of Api was most effective than other APD with $IC_{50}$ values of $3.69{\pm}0.77{\mu}M$. And the NOI activity was declined in the following order: Api-BG ($IC_{50}=8.91{\pm}1.18{\mu}M$), Api-PG ($IC_{50}=13.52{\pm}0.85{\mu}M$) and API-G ($IC_{50}=17.30{\pm}0.66{\mu}M$). The NOI activity of two novel compounds, Api-PG and Api-BG were lower than their aglycone; Api, but more effective than Api-G (NOI: Api-PG and Api-BG). And their suppression ability on INF cytokines such as $TNF-{\alpha}$, $IL-1{\beta}$ and IL-6 mRNA showed the similar tendency. Therefore, the anti-INF mechanism study of Api-PG and Api-BG on nuclear factor-kappa B ($NF-{\kappa}B$) pathway, representative INF mechanism, was investigated and Api was used as positive control. Api-BF was more effectively prevent the than phosphorylation of $pI{\kappa}B$ kinase (p-IKK) and p65 than Api-PG in Raw 264.7 cells. In contrast, Api-PG and Api-BG were not reduced the phosphorylation of inhibitor of kappa B alpha ($I{\kappa}B{\alpha}$). Moreover, pretreatment with Api-PG and Api-BG, dose-dependently inhibited LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNAs and proteins in macrophage cells, and their expression were correlated with their NOI activity. Therefore, APL can be utilized to health promote agent associated with their AIN metabolites.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.3
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• pp.470-486
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• 1995

Mammary epithelial cells contain a subpopulation of cells with a large proliferativ potential which are responsible for the maintenance of glandular cellularity and are the progenitor cells of mammary cancer. These clonogens give rise to multicellular clonal alveolar or ductal units(AU or DU) on transplantation and hormonal stimulation. To isolate putative mammary clonogens, enzymatically monodispersed rat mammary epithelial cells from organoid cultures and from intact glands are sorted by flow cytometry according to their affinity for FITC labeled peanut lectin(PNA) and PE labeled anti-Thy-1.1 antibody(Thy-1.1) into four subpopulations : cells negative to both PNA and Thy-1.1(B-), PNA+cells, Thy-1.1+cells, and cells positive to both reagents(B+). The in vivo transplantation assays indicate that the clonogenic fractions of PNA+cells from out-growths of organoids in primary cultures for three days in complete hormone medium(CHM) are significantly higher than those of cells from other subpopulations derived from cultrues or from intact glands. Extracellular matrix(ECM) is a complex of several proteins that regulated cell function ; its role in cell growth and differentiation and tissue-specific gene expression. It can act as a positive as well as a negative regulator of cellular differentiation depending on the cell type and the genes studied. Regulation by ECM is closely interrelated with the action of other regulators of cellular function, such as growth factors and hormones. Matrigel supports the growth and development of several different multicellular colonies from mammary organoids and from monodispersed epithelial cells in culture. Several types of colonies are observed including stellate colonies, duct-like structures, two- and three-dimensional web structures, squamous organoids, and lobulo-duct colonies. Organoids have the greatest proliferative potential and formation of multi-cellular structures. Phase contrast micrographs demonstrate extensive intracellular lipid accumulation within the web structures and some of duct-like colonies. At the immunocytochemical and electron micrograph level, casein proteins are predominantly localized near the apical surface of the cells or in the lumen of duct-like or lobulo-duct colonies. Squamous colonies are comprised of several layers of squamous epithelium surrounding keratin pearls as is typical fo squamous metaplasia(SM). All-trans retinoic acid(RA) inhibits the growth of SM. The frequency of lobulo-ductal colony formation increased with the augmentation of RA concentration in these culture conditions. The current study models could provide powerful tools not only for understanding cell growth and differentiation of epithelial cells, but also for the isolation and characterization of mammary clonogenic stem cells.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.4
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• pp.517-524
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• 2011

Eleven polyphenolic compounds, including procatechuic and chlorogenic acids, (+)-dihydroquercetin, rutin, isoquercitrin, quercitrin, (+)-dihydrokaempferol, trans-resveratrol, moracin, quercetin and 4-prenylmoracin were isolated and purified from the methanolic extract of defatted mulberry seed residue by a series of column chromatography including silica gel, Sephadex LH-20, and ODS-A, and their chemical structures were identified by spectral analysis. The antioxidant activities of the eleven isolated polyphenolic compounds were measured spectrophotometrically using DPPH radical. Among the eleven polyphenolic compounds tested, rutin ($IC_{50}=20.2\;{\mu}M$), isoquercitrin ($IC_{50}=22.5\;{\mu}M$), quercitrin ($IC_{50}=24.6\;{\mu}M$), quercetin ($IC_{50}=27.8\;{\mu}M$), (+)-dihydroquercetin ($IC_{50}=28.9\;{\mu}M$), and chlorogenic acid ($IC_{50}=30.6\;{\mu}M$) exhibited stronger antioxidant activity than L-ascorbic acid ($IC_{50}=31.5\;{\mu}M$) and ${\alpha}$-tocopherol ($IC_{50}=52.3\;{\mu}M$), whereas procatechuic acid ($IC_{50}=68.2\;{\mu}M$) showed lower activity. In addition, (+)-dihydrokaempferol ($IC_{50}=33.8\;{\mu}M$), trans-resveratrol ($IC_{50}=36.2\;{\mu}M$), moracin ($IC_{50}=47.6\;{\mu}M$), and 4-prenylmoracin ($IC_{50}=48.2\;{\mu}M$) exhibited moderate antioxidant activity. Furthermore, levels of the eleven polyphenolic compounds from three different types of mulberry seeds were quantified by HPLC, and their contents were as follows: rutin (311~60.0 mg/100 g)> quercitrin (7.2~34.2 mg/100 g)> (+)-dihydroquercetin (13.2~33.1 mg/100 g)> quercetin (15.8~19.5 mg/100 g)> 4-prenylmoracin (10.5~43.3 mg/100 g)> isoquercitrin (5.8~15.4 mg/100 g)> chlorogenic acid (0.0~15.3 mg/100 g)> moracin (4.7~7.2 mg/100 g)> procatechuic acid (0.0~11.6 mg/100 g)> (+)-dihydrokaempferol and trans-resveratrol (<0.1 mg/100 g). The 'Daesungppong' mulberry seeds among the three cultivars had higher flavonoid contents, such as rutin and quercetin derivatives, while the 'Iksuppong' seeds had the highest contents of phenolic acids and moracin derivatives. 'Cheongilppong' had lower amounts of polyphenolic compounds than the other two mulberry seeds. These results indicate that mulberry seeds containing antioxidant polyphenolic compounds may be potentially useful sources of anti-diabetic, anti-hypertensive, and anti-aging agents for functional foods and cosmetics.

• Food Science and Preservation
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• v.15 no.3
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• pp.483-490
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• 2008

Several tartaric acid-degrading bacteria were isolated from Korean grape wine pomace after enrichment culture at $30^{\circ}C$ for 10 days in liquid media containing tartaric acid Among them, strains KMBL 5777 and KMBL 5778 exhibited the highest level in the growth and tartaric acid degradability in a medium containing 0.2%(w/v) tartaric acid as a sole carbon source. They were identified as Acetobacter tropicalis based on their morphological and physiological characteristics as well as their 16S rDNA sequences. Blast search of the 16S rDNA sequences revealed that the isolated strains are closest to Acetobacter tropicalis. Homologies of the sequences of KMBL 5777 and KMBL 5778 were 96.0 and 98.9%, respectively with those of A. tropicalis LMG 1663. Both the two bacteria showed higher tartaric acid degradation at $25^{\circ}C$ that those at 20 and $30^{\circ}C$. They could degrade tartaric acid at a wide range of pH between 4.0 and 7.0 with the most rapid degradability at pH 7.0. However, when the bacteria were grown for 8 days, the same level of tartaric acid degradation was observed at pH 4.0, 5.0, 6.0 and 7.0, which was 90.0% of degradation of the acid.

• Food Science and Preservation
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• v.24 no.8
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• pp.1168-1179
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• 2017

In this study, we tried to screen the Bacillus strain having safety probability by isolation of strains from traditional fermented food, measurement of probiotic properties, and the fermentative characteristics of Cheonggukjang. We isolated 400 Bacillus-like isolates from traditional fermented foods. Selected strains examined on the prevalent characteristic such as extracellular enzyme and antibacterial activities, and their safety probability was confirmed by biogenic amine productivity, hemolytic, and harmful substances and enzyme productivity. We selected the 5 strains by analysis of biogenic amine, antibacterial and B. cereus toxic associated gene. Five selected strains were examined on cell surface hydrophobicity, and bile and acid tolerance, and we selected the SRCM100730 as the final strain. SRCM100730 was confirmed B. amyloliquefaciens by 16S rRNA sequencing, and named the B. amyloloquefaciens SRCM100730 (KCCM11966P). Finally, we manufactured Cheonggukjang using SRCM100730 for confirmation of fermentation properties. Manufactured Cheonggukjang did not contain B. cereus, and showed that ${\gamma}$-PGA and extracellular enzyme activities were superior to commercial Chunggukjang. Amino nitrogen content was 544.02 mg% and 26 free amino acid were detected, and the bitterness-related amino acid content was lower than commercial Cheonggukjang. Especially, the amount of GABA was 3 fold higher than commercial Cheonggukjang. These results suggest that SRCM100730 have high availability in commercial probiotics market and fermented food industry.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.3
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• pp.186-196
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• 2010

Introduction: The first aim of this study was to isolate the dental tissue-derived stem cells from the dental follicle (DF), dental pulp (DP), and root apical papilla (RAP) of the extracted wisdom teeth. Second was to evaluate their characterization with the expressions of transcription factors and cell surface markers. Finally, their ability of the in vitro multi-lineage differentiations into osteogenic and adipogenic cells were compared, respectively. Materials and Methods: Dental tissues, including dental follicle, dental pulp, and root apical papilla, were separated in the extracted wisdom teeth. These three dental tissues were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with supplements, respectively. After passage 3, the homogeneous shaped dental tissue-derived cells were analyzed the expression of transcription factors (Oct-4, Nanog and Sox-2) and cell surface markers (CD44, CD90 and CD105) with reverse transcription polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. In order to evaluate in vitro multi-lineage differentiations, the culture media were changed to the osteogenic and adipogenic induction mediums when the dental tissue-derived cells reached to passage 3. The characteristics of these three dental tissue-derived cells were compared with immunohistochemistry. Results: During primary culture, heterogenous and colony formatted dental tissue-derived cells were observed in the culture plates. After passage 2 or 3, homogenous spindle-like cells were observed in all culture plates. Transcription factors and mesenchymal stem cell markers were positively observed in all three types of dental tissue-derived cells. However, the quantity of expressed transcription factors was most large in RAP-derived cells. In all three types of dental tissue-derived cells, osteogenic and adipogenic differentiations were observed after treatment of specific induction media. In vitro adipogenic differentiation was similar among these three types of cells. In vitro osteogenic differentiation was most strongly and frequently observed in the RAP-derived cells, whereas rarely osteogenic differentiation was observed in the DP-derived cells. Conclusion: These findings suggest that three types of human dental tissue-derived cells from extracted wisdom teeth were multipotent mesenchymal stem cells, have the properties of multi-lineage differentiations. Especially, stem cells from root apical papilla (SCAP) have much advantage in osteogenic differentiation, whereas dental follicle cells (DFCs) have a characteristic of easy adipogenic differentiation.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.235-243
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• 2000

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.

• Clinical and Experimental Reproductive Medicine
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• v.11 no.1
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• pp.17-32
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• 1984

There are numerous reports on the relative risk of pelvic inflammatory disease among the users versus the nonusers of intrauterine device. Reported relative risk varied from no difference between the two groups to 3-9 fold increase in the users. In an attempt to define this relative risk of pelvic inflammatory disease and related microorganisms ,pelvic organ observation and bacteriological study were done through laparoscopy. Specimens for microbiologic culture were obtained simultaneously from the fallopian tubes via laparoscopy and from the endocervix via regular pelvic examination method. The study population was consisted of 30 I.U.D.users and 35 J.U.D.nonusers who visited the Yonsei University Severance Hospital and the Sung-Ga Hospital for laparoscopic sterilization. The results obtained were as follows: 1. There was no difference in age distribution, economic status and numbers of parity and abortion between I.U.D. users and I.U.D. nonusers. 2. The pelvic inflammatory findings were noted on laparoscopy in 2 cases of I.U.D. users, with an incidence of 6.6%. And no pelvic inflammatory finding was noted in any of the nonusers,but this difference was not statistically significant (p>0.005). 3. All the bacteriologic culture of the specimens from the fallopian tubes of both groups yielded negative results. 4. The bacteriologic culture of the spec imens f rom the endocervix revealed more frequent isolation of possible pathogen such as Hem ophilus ,alpha-Streptococcus ,Corynebacteria, Bacteroides in the I.U.D.users than in the nonusers.But,this difference was also not statistically significant (p>0.005).

• Journal of Life Science
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• v.21 no.12
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• pp.1666-1677
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• 2011

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.