• Restorative Dentistry and Endodontics
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• v.27 no.5
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• pp.463-472
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• 2002

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-$\alpha$ from immune cells. Although rnonocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-$\alpha$. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)$_2$ may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-$\alpha$, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 $\mu\textrm{g}$/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)$_2$ at 37$^{\circ}C$ for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4$\times$10$^6$ cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10$\mu\textrm{g}$/ml) for 24 hours at 37$^{\circ}C$ in 5% $CO_2$ incubator. The supernatants of cells were collected and the levels of IL-1$\alpha$, IL=1$\beta$ and TNF-$\alpha$ were measured by enzyme-linked immunosorbent assay. The results were as follows ; 1. The levels of IL-1$\alpha$, IL-1$\beta$, TNF-$\alpha$ from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p<0.05). 2. The levels of all three cytokines released from PMN stimulated with each calcium hydroxide treated LPS were significantly lower than those released from PMN stimulated with each untreated LPS (p<0.05), while they were not significantly different from those released from unstimulated PMN of the control group (p>0.05) 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p<0.05), but not in PMN stimulated with each calcium hydroxide treated LPS (p>0.05). 4. The levels of all three cytokines released from PMN stimulated with p. endodontalis LPS were significantly lower than those released from PMN stimulated with E coli LPS (p<0.05).

• Restorative Dentistry and Endodontics
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• v.32 no.2
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• pp.121-129
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• 2007

The purpose of this study was to assess the antibacterial effect of sodium dichloroisocyanurate (NaDCC), sodium hypochlorite (NaOCl), and chlorhexidine (CHX) on Enterococcus faecalis and to evaluate and to compare the time-dependant antimicrobial effect of NaDCC with NaOCl and CHX in the root canal in vitro before and after instrumentation. Extracted Human single teeth were prepared by serial instrumentation technique. The samples were autoclaved and contaminated for 3 days with E. faecalis monocultures. The teeth were then divided into 4 groups Each group was irrigated and inserted with 2% NaOCl, 2% NaDCC, 2% CHX and steri)ized saline. After 6, 12, 24, 72h, and 1 week incubation, sterilized paper point was inserted into the root canal. Paper points containing root canal contents were then placed on the agar plate. And then each root cana) was prepared with #4 and #5 GG (Gates-Glidden) drill. The debris were collected in the sterilized microtube and the plates were incubated at $37^{\circ}C$ in an increased $CO_2$ atmosphere. After 24h incubation the growth of bacteria around the paper points were measured. NaOCl and NaDCC solution shows similar antimicrobial effect for E. faecalis at 6, 12, 24, 72h and 1 week. In centrol group, irrigated with sterilized saline, no antimicrobial effect was observed. The results are in agreement with other investigators, who have shown the bactericidal property and possibility of NaDCC as a root canal irrigation solution. Thus it seems that NaDCC solutions can be clinically applied into the root canal within 1 week after dilution.

• Applied Microscopy
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• v.7 no.1
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• pp.21-43
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• 1977

This experiment was undertaken in order to localize the labeled dbcAMP (dibutyryl cyclic AMP) in oocytes whose development has been suppressed by cold dbcAMP for 6 or 19 hours in vitro. Mouse oocytes were obtained from the ovaries of 3-4 week old A strain female mice, by puncturing the Graafian follicles in the modified Krebs-Ringer bicarbonate salt solution under the dissecting microscope. Those oocytes which have intact germinal vesicle were cultured in the basic culture medium supplemented with 0.4% bovine serum albumin (BSA). Cultivation of the oocytes was carried out in a microtube developed by Cho (1974). The cultures were then incubated in a humidified 5% $CO_2$ incubator maintained at $37^{\circ}C$ for 6 or 19 hours (Donahue, 1968). DbcAMP was added to culture medium for a final concentration of 100ug/ml, and $^3H-dbc$ AMP (specific activity 13 Ci/mM) for a final concentration of $40{\mu}Ci/ml$ was also added to the medium. For electron microscopic autoradiography, those oocytes recovered from the culture were washed with phosphate buffer (pH 7.4), and immediately prefixed in a 2.5% glutaraldehyde overnight and postfixed for 2 hours at $4 ^{\circ}C$ in 1% osmium tetroxide in phosphate buffer with pH 7.4 (Palade, 1952). After fixation, the materials were dehydrated in graded alcohol series and embedded in Epon 812 mixture based on the standard procedures (Luft, 1961). The thin sections $600-700{\AA}$ thick were mounted on the grids of 200 meshes. The grids containing sections were coated with a nuclear emulsion Kodak NTB-3 and stored in a cold dark box (at $4^{\circ}C$) for 3 weeks. After exposure, the samples were developed with Kodak D-19 and stained with uranyl acetate and lead citrate. Routine observation was made with Hitachi HU-11E electron microsocope. The results of the observation were as followings: 1. It was found that the labeled dbcAMP penetrated the egg plasma membrane and dispersed at random in the cytoplasm. 2. It was also observed that most of the labeled dbcAMP was attached to microfibrillar lattices portion of the oocyte cytoplasm. There fore, it is presumed that the receptor of the dbcAMP is localized in the microfibrillar lattices of the oocyte. 3. It also seems that some other cell organells such as mitochondria, Golgi complex, cortical granules are not directly related to the action of the dbcAMP. 4. The labeled dbcAMP was neither observed in the membrane nor in the nucleus. Therefore, it seems that there is no relationship between the concentration of dbcAMP and the nuclear membranous permeability. 5. There was no difference in number of dbcAMP particles when oocytes were cultured for 6 hours and 19 hours. 6. However, it was observed that, in same of the oocytes suppressed in germinal vesicle by dbcAMP for 19 hours, cell organells were moved and concentrated to a small portion of the cytoplasm, and that the morphology of the organells greatly changed to an abnormal. form. Therefore, it is supposed that those oocytes were in the process of degeneration. From the above results, it is expected that dbcAMP penetrated the egg membrane and was bound to the receptor which seems to be located in the microfibrillar lattiees portion, and that this dbcAMP-receptor complex inhibited some enzyme system of the oocytes which are essential for the germinal vesicle breakdown.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.79-87
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• 2005

The ovaries from Hanwoo and Holstein were collected from labattoir and transferred to laboratory. Oocytes were aspirated and incubated in $CO_2$ incubator for 24 hours for maturation. Oocytes were coincubated with the sperms for 5 hours. Cleaved oocytes were selected 48 hours after coincubation and half of the medium was changed newly every 48 hour until blastocyst formation. Cleavage rate and blastocyst rate were investigated according to different breeds and different status of cumulus cells surrounding the oocytes. Blastocysts were either transferred to the recipients or frozen until use. The result of embryo transfer with fresh or frozen embryos was investigated. The rate of male offspring following embryo transfer was also investigated. The rate of cleavage was $66.4\%$ for Hanwoo and $62.4\%$ for Holstein oocytes. The rate of cleavage according to status of oocyte was shown highest in the oocytes completely surrounded with cumulus cells and lowest in denuded oocytes for both Hanwoo and Holstein oocytes. The rate of blastocyst from cleaved oocytes was $40.6\%$ for Hanwoo and $36.9\%$ for Holstein. The rate of pregnancy/delivery following embryo transfer with fresh IVF embryos was $57.2\%$ for Hanwoo and $53.3\%$ for Holstein. The rate of pregnancy/delivery following embryo transfer with frozen IVF embryos was $40.9\%$ for Hanwoo and $36.4\%$ for Holstein. The rate of male calf produced by embryo transfer was $63.6\%$ for Hanwoo and $50.0\%$ for Holstein.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.23 no.4
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• pp.289-296
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• 1990

The antimicrobial and antioxidant activities of grapefruit seed extract(GFSE), which was extracted with glycerine in the special schematic extraction apparatus, were investigated for handling and processing of fishery products. The effectivity of GFSE has been tried on sardine, mackerel and shrimp divided into six lots for each fishery product: control(no treatment) and five GFSE-treated samples. Samples were inoculated with Salmenella typhi, incubated for 24hrs at $30^{\circ}C$ in dextrose-tryptone broth medium and prepared for microbiological 8f chemical analysis and organoleptic assessment. The bacteriological analytical results with GFSE(250ppm) showed the reduction of $1.8\times10^6\to2.0\times10^4,\;1.9\times10^6\to1.8\times10^4$ and $1.6\times10^6\to2.7\times10^3$ in total bacterial count for sardine, mackerel and shrimp, respectively. The test results with GFSE(500ppm) showed a $100\%$ reduction of bacterial mackerel treated with GFSE(500ppm) was reduced to $1.1\times10^4$ and $9.0\times10^3$ respectively. Antioxidant effect of treatment with GFSE at 500ppm level for three products was significant. LSD test results on organoleptic parameter for the samples treated with various showed a significant influence on the appearance, odor and texture in which at concentration 500ppm level give the excellent scours compared to each control.

• Tuberculosis and Respiratory Diseases
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• v.49 no.2
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• pp.217-224
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• 2000

Background : It is well known that when macrophages are stimulated with endotoxin, they produce a wide variety of cytokine mediators, including TNF-$\alpha$ and IL-1$\beta$. However, there is an alteration in the macrophages' responsiveness when they are challenged with repeated bouts of endotoxin, termed "endotoxin tolerance" which is regarded as a self-protective phenomenon from continuous stimulation. In this study, endotoxin tolerance in the peripheral blood monocytes of sepsis patients was evaluated. Methods : Fourteen patients with organism-documented sepsis were included. The severity of illness was evaluated by APACHE II score. Peripheral blood monocytes were isolated from the patients and diluted to $1{\times}10^5$ well. After stimulation with endotoxin (LPS of E. coli O114 : B4, 100 ng/ml), they were incubated at $37^{\circ}C$ in 5% $CO_2$ incubator for 24 hours. Supernatant was collected for the measurement of TNF-$\alpha$ and IL-1$\beta$ with ELISA method. Peripheral blood monocytes of seven healthy volunteers were used as control. Results : The APACHE II score (mean$\pm$SD) of the patients at the time of blood sampling was 12.2$\pm$5.7. The primary infection foci were urinary tract infection, pneumonia, subacute bacterial endocarditis, and catheter related infection, etc. The causative organisms were gram negative rods (10 cases), gram positive cocci (6 cases) with two cases of mixed infection. Serum TNF-$\alpha$ could be measured in 4 cases with 29.9$\pm$27.7 pg/ml. Serum IL-1$\beta$was measurable in only one patient. The TNF-$\alpha$ level of supernatant of cultured peripheral blood monocytes was 2,703$\pm$2,066 pg/ml in patients and 2,102$\pm$1914 pg/ml in controls. The IL-1$\beta$level of supernatant was 884$\pm$1,050 pg/ml in patients and 575$\pm$558 pg/ml in controls. There was no difference of TNF-$\alpha$ and IL-1$\beta$ level between patients and controls. Conclusion : We cannot prove the phenomenon of endotoxin tolerance in this study. Future study needs to be focused on the more severe sepsis patients who were taken for sampling earlier. Addition of serum to the culture medium could be an another valuable option for the success of this study.

• 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.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.332-338
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• 2009

A xylan-decomposing bacterium, HY-20, was isolated from the gut of a honeybee, Apis mellifera, and identified as Bacillus sp. The extracellular GH11 xylanase (XylP) gene (687-bp) of strain HY-20 encoded a protein of 228 amino acids with a deduced molecular mass of 25,522 Da and a calculated pI of 9.33. The primary structure of XylP was 97% identical to that of B. pumilus xylanase (GenBank accession no.: AY526092) that has not been characterized yet. The recombinant His-tagged enzyme (rXylP) overexpressed in Escherichia coli BL21 harboring pET-28a(+)/xylP was purified to electrophoretic homogeneity by cation exchange and gel permeation chromatographies. The purified enzyme exhibited the highest catalytic activity toward birchwood xylan at pH 6.5 and $50^{\circ}C$ and retained approximately 50% of its original activity when pre-incubated at $55^{\circ}C$ for 15 min. The recombinant enzyme was completely inactivated by $Hg^{2+}$ (1 mM) and N-bromosuccinimide (5 mM), while its activity was slightly stimulated by approximately 10% in the presence of $Mn^{2+}$ (1 mM), $Fe^{2+}$ (1 mM), and sodium azide (5 mM). rXylP was able to efficiently degrade various polymeric xylose-based substrates but PNP-sugar derivatives and glucose-based polymers were not susceptible to the enzyme.

• KSBB Journal
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• v.21 no.1 s.96
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• pp.72-78
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• 2006

This study was carried out to investigate the antimicrobial and antioxidative effects of mycelium cultural extract from mushroom. Mushroom mycelium was grown in a defined synthetic liquid medium and citrus extracts, and the culture extracts were examined for antioxidant and antibacterial activity. Myceliums of Phellinus linteus, Cordyceps militaris, Coriolus versicolor, Sparassic crispa, Agaricus blazei, lnonotus obliquus, Lentinus edodes, Hericium erinacium, Gonoderma lucidium in 10% citrus extract supplemented medium and synthesis medium were incubated in a shaking incubator (120 rpm, $24{\sim}30^{\circ}C$ ) for $7{\sim}15$ days. The antimicrobial activity of the culture fluid of mushroom mycelium grown in submerged liquid culture was tested against 12 microorganisms which were fish pathogens and common bacterial species. The culture extracts showed high activity against Vibrio sp. and had poor effect on Streptocouus sp., S. parauberis, S. iniae. The culture extracts obtained from the synthetic medium showed $30{\sim}93%$ of the 1,1-diphenyl-2-picrylhydrazyl radical scavenger activity, the culture extracts obtained from the citrus extracts medium exhibited antioxidant activity up to 55%.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.11
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• pp.1564-1570
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• 2016

The present study investigated the immunomodulatory effects of high-purity ${\beta}$-1.3/1.6-glucan on macrophages, natural killer (NK) cells, and T cell-mediated factors. Effect of high-purity ${\beta}$-1.3/1.6-glucan on cytotoxicity in macrophages was investigated. Using macrophages, cytotoxicity of high-purity ${\beta}$-1.3/1.6-glucan was evaluated by MTT assay. We treated high-purity ${\beta}$-1.3/1.6-glucan at concentrations of 10, 50, 100, 150, 200, and $250{\mu}g/mL$ in macrophages. High-purity ${\beta}$-1.3/1.6-glucan did not affect macrophage viability. Phagocytic activity was assessed using zymosan. Activity of high-purity ${\beta}$-1.3/1.6-glucan on macrophages significantly increased as compared with zymosan. We treated high-purity ${\beta}$-1.3/1.6-glucan to murine NK cells co-incubated with YAC-1 cells. High-purity ${\beta}$-1.3/1.6-glucan resulted in significantly increased activity of NK cells as compared with the control. In addition, treatment of macrophages with high-purity ${\beta}$-1.3/1.6-glucan resulted in significantly increased activity of T cell-mediated cytokine (IL-2, IL-12, $IFN-{\gamma}$, and $TNF-{\alpha}$) levels and CD4+/CD8+ T cells as compared with the control. In conclusion, high-purity ${\beta}$-1.3/1.6-glucan could enhance the immune response through activation of macrophages, NK cells, and T cell-mediated factors.