• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.1 s.49
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• pp.51-58
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• 2005

Alkyl ethoxy sulfate type surfactants, widely used in commercial cleansers, are easily adsorbed to skin to often cause skin irritation and inflammation if not thoroughly rinsed nut. In order to replace or complement existing surfactants, we screened the existing surfactants through protein denaturation method, cell cytotoxicity assay and human IL-1$\alpha$ assay, etc. Fourteen surfactants have been chosen from among too irritant anionic, cationic and/or zwitter-ionic ones and investigated for cell cytotoxicity in human fibroblast cell lines using monolayer culture with the thirteen commercially available cleansers for sensitive skin. From these results, we selected 5 surfactants and 2 commercial cleansers (names not shown), such as sodium laureth sulfate (anionic), sodium cocoyl isethionate (anionic), sodium lauroamphoacetate (zwitter-ionic), and cocamidopropyl betaine (zwitter-ionic), alkyl polyglycoside (non-ionic). 20 formulations were made out of 5 surfactants and five of them were chosen through a protein denaturation method (lower than 3 M sodium dodecyl sulfate solution ($13.2\%$)), cell cytotoxicity and human patch test. These five selected formulations containing preservatives were compared to two selected commercial cleansers by cell cytotoxicity and human IL-1$\alpha$ ELISA assay using dermal equivalent. Finally, we selected the best formulation. To this formulation, fructan ($3\%$ or $5\%$) or/and portulaca extract ($3\%$ or $5\%$) well known for its anti-inflammatory and moisturizing effects were added and investigated for cell cytotoxicity using dermal equivalent. In cytotoxicity assay using dermal equivalent, two formulations containing $5\%$ fructan and $3\%$ or $5\%$ portulaca extract were less toxic than the others. In cytotoxicity assay and human IL-1$\alpha$ ELISA using 3D culture, the selected formulation containing $5\%$ fructan and $5\%$ portulaca extract showed better efficiency than those of the others and 2 commercial cleansers. As a result, we could develop a low irritant and safe liquid cleanser.

• Tuberculosis and Respiratory Diseases
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• v.40 no.3
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• pp.236-249
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• 1993

Background: Among the injurious agents to which the lung airspaces are constantly exposed are reactive species of oxygen. It has been widely believed that reactive oxygen species may be implicated in the etiology of lung injuries. In order to elucidated how this oxidant causes lung cell injury, we investigated the effects of exogenous $H_2O_2$ on alveolar epithelial barrier characteristics. Methods: Rat type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with HEPES-buffered Ringer solution. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against $H_2O_2$ injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ, an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results: These monolayers have a high transepithelial resistance (>2000 ohm-$cm^2$) and actively transport $Na^+$ from apical fluid. $H_2O_2$(0-100 mM) was then delivered to either apical or basolateral fluid. Resulting indicated that $H_2O_2$ decreased Isc and R gradually in dose-dependent manner. The effective concentration of apical $H_2O_2$ at which Isc (or R) was decreased by 50% at one hour ($ED_{50}$) was about 4 mM. However, basolateral $H_2O_2$ exposure led to $ED_{50}$ for Isc (and R) of about 0.04 mM. Inhibition of cellular catalase yielded $ED_{50}$ for Isc (and R) of about 0.4 mM when $H_2O_2$ was given apically, while $ED_{50}$ for basolateral exposure to $H_2O_2$ did not change in the presence of ATAZ. The rate of $H_2O_2$ consumption in apical and basolateral bathing fluids was the same, while cellualr catalase activity rose gradually with time in culture. Conclusion: Our data suggest that basolateral $H_2O_2$ may affect directly membrane component (e.g., $Na^+,\;K^+$-ATPase) located on the basolateral cell surface. Apical $H_2O_2$, on the other hand, may be largely degraded by catalase as it passes through the cells before reaching these membrane components. We conclude that alveolar epithelial barrier integrity as measured by Isc and R are compromised by $H_2O_2$ being relatively sensitive to basolateral (and insensitive to apical) $H_2O_2$.

• Clinical and Experimental Pediatrics
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• v.50 no.12
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• pp.1194-1199
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• 2007

Purpose : It was generally accepted now a days that the pathogenesis of preeclampsia, small for gestational age (SGA), intrauterine growth retardation and fetal origin of adult diseases were related with a endothelial cell dysfunction. The purpose of this study was to know the relation of such diseases by assessing the level of endothelin-1. Methods : SGA babies, newborns of preeclampsia and normal control mother were included in this study. Isolated endothelial cells were centrifugated and mixed with media in $37^{\circ}C$, 5% $CO_2$ to obtain confluent monolayer of cultured human umbilical venous endothelial cell (HUVEC). Endothelin-1 levels were determined by Endothelin-1 colorimetric (EIA) Kits. We examined the endothelin-1 level in the HUVEC supernatants from SGA baby, and newborns from preeclampsia as well as normal mother. Also, we compared the endothelin-1 level of cultured normal HUVEC incubated with serum from cord blood of SGA, babies of preeclampsia or normal control mother. Results : The endothelin-1 levels in cultured HUVEC supernatants of three groups showed no significant difference but the endothelin-1 levels of cultured normal HUVEC incubated with serum from preeclampsia mother or SGA mother was significantly higher than those from newborns of control mothers (P<0.05). Conclusion : These findings suggest that there may be the factor which affect the endothelin-1 level in serum of cord blood from SGA and preeclampsia.

• Parasites, Hosts and Diseases
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• v.27 no.2
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• pp.87-100
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• 1989

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that 2. tenezla first penetrate into the mucosal intraepithelial Iymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope labelled uracil ($^3H-uracil$) . Third, the E. tenella sporozoites viability was assayed after preincubation of them with thicken spleen cells. E. tenella oocysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (I) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schisogonic cycle of E. tenella in 3~4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merogoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48~60 hours, and decreased thereafter. The uptake amount of $^3H-uracil$ depended not only upon the inoculum sixte of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.

• Korean Journal of Environmental Agriculture
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• v.24 no.2
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• pp.146-152
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• 2005

The objective of present study was to investigate the effect of onion extracts on mercuryinduced cytotoxicity, lipid peroxidation and antioxidant enzyme activities in primary monolayer cultures of rat hepatocytes. Primary cultures of rat hepatocytes were incubated for 6 hr in the presence of various concentrations (0, 1, 5, 10, 30 or 50 ppm) of $HgCl_2$. Cytotoxicity and cell viability were determined by measuring glutamic oxaloacetic transaminase (GOT) activity, lactate dehydrogenase (LDH) activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) value. Lipid peroxidation w as evaluated using thiobarbituric acid reactive substances (TBARS) assay. Effects of onion extract on antioxidant system were determined by measuring catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) activities as well as DPPH free radical scavenging activity. $HgCl_2$ at the concentration of 10 ppm increased GOT activity and TBARS concentration but decreased %MTT reduction, whereas $HgCl_2$ at the concentration of 30 ppm increased LDH activity, representing that $HgCl_2$ caused cytotoxicity and lipid peroxidation in dose-dependent manner, $HgCl_2$ at the concentration of 30 ppm significantly decreased catalase, GSH-Px and GSH-Rd activities. When primary cultures of rat hepatocytes were incubated with various concentrations (0, 0.01, 0.05, 0.1 or 0.3 mg/ml) of onion extract for 6 hr in the presence of 30 ppm of $HgCl_2$, onion extracts at the concentration of 0.05 mg/ml decreased GOT activity, but increased %MTT reduction by 30 ppm of $HgCl_2$. $HgCl_2-induced$ LDH activity and TBARS concentration were decreased by onion extract at the concentration of 0.01 mg/ml. Taken together, onion extract prevented H$HgCl_2-induced$ hepatocyte injury and lipid peroxidation. Onion extracts at the concentration of 0.1 mg/ml almost or completely inhibited $HgCl_2-induced$ catalase and GSB-Px activities. GSH-Rd activity, however, was not affected by onion extract. Free radical scavengjing activity was increased as concentration of onion extract increased. Onion extract at the concentrion of 5 mg/ml possesed mote than 93% scavenging activity comparing to 100% radical scavenging activity by pyrogallol solution as a reference. These results demonstrate that onion extracts suppressed mercury-induced cytoctoxicity and lipid peroxidation by scavenging free radical and increasing catalase and GSH-Px activities.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.9-20
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• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• Tuberculosis and Respiratory Diseases
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• v.43 no.2
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• pp.210-220
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• 1996

Background : Neutrophils or monocytes separated in vitro by the adherence to plastic surface are known to be activated by surface adherence itself and subsequent experimental data might be altered by surface adherence. In the process of surface adherence, adhesion molecules have a clear role in intracellular signal pathway of cellular activation. Human alveolar macrophages(HAM) are frequently purified by the adherence procedure after bronchoalveolar lavage. But the experimental data of many reports about alveolar macrophages have ignored the possibility of adhesion-induced cellular activation. Method : Bronchoalveolar lavage was performed in the person whose lung of either side was confirmed to be normal by chest CT. With the measurement of hydrogen peroxide release from adherent HAM to plastic surface and non-adherent HAM with or without additional stimulation of phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), we observed the effect of the adherence to plastic surface. We also evaluated the effect of various biological surfaces on adhesion-induced activation of HAM. Then, to define the intracellular pathway of signal transduction, pretreatment with cycloheximide, pertussis toxin and anti-CD11/CD18 monoclonal antibody was done and we measured hydrogen peroxide in the culture supernatant of HAM. Results : 1) The adherence itself to plastic surface directly stimulated hydrogen peroxide release from human alveolar macrophages and chemical stimuli such as phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine(fMLP) colud not increase hydrogen peroxide release in these adherent macrophages which is already activated. 2) PMA activated human alveolar macrophages irrespective of the state of adhesion. However, fMLP stimulated the release of hydrogen peroxide from the adherent macrophages, but not from the non-adherent macrophages. 3) HAM adherent to A549 cell(type II alveolar epithelium-like human cell line) monolayer released more hydrogen peroxide in response to both PMA and fMLP. This adherence-dependent effect of fMLP was blocked by pretreatment of macrophages with cycloheximide, pertussis toxin and anti-CD18 monoclonal antibody, Conclusion : These results suggest that the stimulatory effect of PMA and fMLP can not be found in adherent macrophage because of the activation of human alveolar macrophage by the adherence to plastic surface and the cells adhered to biologic surface such as alveolar epithelial cells are appropriately responsive to these stimuli. It is also likely that the effect of fMLP on the adherent macrophage requires new protein synthesis via G protein pathway and is dependent on the adhesion between alveolar macrophages and alveolar epithelial cells by virtue of CD11/CD18 adhesion molecules.