• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• Journal of Life Science
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• v.23 no.9
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• pp.1147-1154
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• 2013

This study was conducted to evaluate the antioxidation activity of Jeju crossbred horse (Jeju native horse ${\times}$ thoroughbred) leg bone extracts (HLBE) and its enzyme hydrolysates by determination of 1,1-diphenyl-2picryl-hydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activity, ferric reducing/antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). HLBE was extracted with hot water for 24 hr and lyophilized. The lyophilized HLBE was hydrolyzed using multifect PR6L (MP), pepsin (PS), and a pepsin and pancreatin mixture (PSPC) for 4 hr at 60, 50, and $50^{\circ}C$, respectively. The hydrolysates were separated by a molecular weight of 3 kDa more or less. When the yield of HLBE was 100%, the yield of hydrolysates less than 3 kDa of MP, PS, and PSPC was 10.86, 3.26, and 8.00%, respectively. Enzyme hydrolysates with low molecular weight less than 3 kDa in MP and PSPC showed significantly higher DPPH, ABTS radical scavenging activity, and ORAC compared to HLBE and its hydrolysates with more than 3 kDa. However, the FRAP of the hydrolysates less than 3 kDa in PS was significantly higher than in MP. These results suggest that low molecular weight enzyme hydrolysates less than 3 kDa have more powerful antioxidation activity, especially when they are hydrolyzed by MP and PSPC rather than PS. Therefore, low molecular weight enzyme hydrolysates of HLBE hydrolyzed with MP and PSPC have significant potential as antioxidants in the food industry. Further in vivo studies are required to support the antioxidation activities of the hydrolysates in vitro.

• Korean Journal of Environmental Biology
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• v.35 no.2
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• pp.116-122
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• 2017

Basic dyes such as malachite green and methylene blue have been used as disinfectants to control water fungal infections since the 1930s. However, after succeeding reports of carcinogenicity and bioaccumulation of the dye, their use was forbidden in lieu of public health. This study undertook to evaluate the therapeutic effect of sulfur solution processed by effective microorganisms (EM-PSS) against Saprolegnia parasitica infection, and its safety in fish. In vitro antifungal evaluation of EM-PSS inhibited the growth of S. parasitica mycelia at concentrations of 50 ppm or higher. The acute toxicity test of EM-PSS to the mud fish (Misgurnus mizolepis) measured a no effect concentration (NOEC) at 100 ppm, the lowest effect concentration (LOEC) at 125 ppm, and the half-lethal concentration ($LC_{50}$) at 125 ppm in juvenile and 250 ppm in the immature stage. In addition, the ecotoxicity test of EM-PSS using Daphnia magna inhibited swimming of D. magna at concentrations of 100 ppm or less. Lastly, the EM-PSS prevented infection of S. parasitica to mud fish, at concentrations of 50 ppm. Furthermore, at 100 ppm concentration, the EM-PSS showed no acute toxicity on mud fish, nor any eco-toxic effects on D. magnano. Therefore, we conclude that carcinogenic disinfectants such as malachite green and methylene blue could be replaced by EM-PSS to remove S. parasitica in mud fish farming, and might be a potential eco-friendly disinfectant in aquaculture.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.235-243
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.1-17
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• 1997

The purpose of this study was to investigate the aspects of proliferation and bone nodule formation of osteogenic precursor cells. To determine the effects of ascorbic acid and dexamethasone upon capacity of osteoblast proliferation and bone nodule formation, cells were maintained in the presence of one or some of these additives for up to 30 days. Group I culture was maintained in standard medium(DMEM plus 10% plus antibiotics), group II was maintained in supplemented medium containing dexamethasone, group III was maintained in supplemented medium containing ascorbic acid and sodium-${\beta}$-glycerophosphate, and group IV was maintained in supplemented containing ascorbic acid, sodium-${\beta}$-glycerophosphate and dexamethasone. Morphology of bone nodules was observed with light microscope and electron microscope. The results were as follows: ${\bullet}$ Proliferation capacity of osteoblasts was not affected by single use of dexamethasone, but it was chiefly affected by ascorbic acid. ${\bullet}$ Cellular morphology was fibroblastic appearance initially, but, it was gradually changed to polygonal shape accompanied by confluency stage. ${\bullet}$ Pluripotent mesenchymal cells existed during primary culture, they were differentiated to adipocyte, chondrocyte, osteocyte according to culture condition. ${\bullet}$ Dexamethasone increased bone nodule formation under the condition that the culture was maintained with supplemented medium ascorbic acid and sodium-${\beta}$-glycerophosphate. ${\bullet}$ when the cultures were stained with alizarin red, the group supplemented with dexamethasone, ascorbic acid and sodium-${\beta}$-glycerophosphate showed the marked increase of bone nodule formation, but the group supplemented with ascorbic acid and sodium-${\beta}$-glycerophosphate revealed only small amounts of bone nodules. And the groups cultured without ascorbic acid showed no observed any of bone-like mass independent of dexamethasone addition.

• Development and Reproduction
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• v.6 no.2
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• pp.105-110
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• 2002

Numerous hormones are involved in the regulation of reproduction. Among them, estrogen and progesterone are the most important ovarian steroid hormones regulating female fertility. On the other hand, diverse stressors impede female receptivity and fertility. Since norepinephrine(NE) and epinephrine(E) are released from the adrenal during stress, it might play a role in stress-induced disruptions of fEmale reproductive parameters. The present study was performed to analyze the changes in adrenal catecholaminergic activities in cycling rats. The tissue content and secretion level of catecholamines were determined by high performance liquid chromatography coupled with electrochemical detector(HPLC-ECD). Adrenomedullary content of norepinephrine(NE) was increased on proestrus stage (59.47 $\pm$ 6.86 ug/gland), peaked on diestrus I stage(65.22 $\pm$ 5.99 ug/gland), and was nadir on diestrus II stage(41.63 $\pm$ 1.33 ug/gland). The highest E content was observed on proestrus stage(361.86 $\pm$ 15.58 ug/gland) while the lowest level was on diestrus II stage(285.58 $\pm$ 12.25 ug/gland). In addition to these observations, a significant reduction of the NE : E ratio was observed (1 : 4.81 on diestrus I vs 1 : 6.13～7.02 on other stages). In vitro secretion of adrenal NE and E was increased on proestrus stage, peaked on estrus stage, and decreased on diestrus II stage. Interestingly, the NE : E ratio in conditioned media was significantly increased on estrus stage (1 : 3.32 vs 1 : 2.34～2.65 on other stages. The biosynthesis of NE and E is mediated by tyrosine hydroxylase(TH) and phenylethanolamine-N-methyltransferase(PNMT) which acts conversion of tyrosine into DOPA and NE into E, respectively. These finding demonstrated that sex steroids, during setrous cycle, seem to be able to modify the adrenal catecholamines biosynthesis and secretion with stage-specific manner by modulation of the enzyme activities.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.11
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• pp.1611-1618
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• 2010

This study was conducted to evaluate the effects of Eriobotrya japonica and Saurus chinensis extracts and their fermented extracts on immune parameters in BALB/c mice treated with 1% 2,4-dinitrochlorobenzene (DNCB). The groups were Eriobotrya japonica extract (BI), Saurus chinensis extract (SA), mixture with E. japonica extract and S. chinensis extract (FB) and fermented mixture with E. japonica extract and S. chinensis extract (FA) and distilled water treated control. The level of serum immunoglobulin (Ig) E was decreased in FA compared to control group, but significant difference was not observed (p<0.05). The histamine and contents in FA and control group were $1.47{\pm}0.20$ ng/mL and $1.90{\pm}0.04$ ng/mL, respectively (p<0.05). Ceramide contents were significantly increased in FA compared to BI, SA, FB and control group (p<0.05). These results suggest that FA supplementation in the DNCB treated BALB/c mice affect anti-allergic activities positively, and may be used as functional material for suppression of atopy dermatitis in food industry.

• Radiation Oncology Journal
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• v.21 no.1
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• pp.75-81
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• 2003

Purpose : This study quantitatively evaluated the apoptosis In human peripheral blood lymphocytes using flow cytometry, and investigated the possibility of using this method, with a small amount of blood, and the time and dose dependence of radiation-induced apoptosls. Materials and Methods : Peripheral blood lymphocyes were isolated from the heparinized venous blood of 11 healthy volunteers, 8 men and 3 women, with each 10 ml of blood being divided Into IS samples. The blood lymphocytes were Irradiated using a linear accelerator at a dose rate of 2.4 Gy/min, to deliver doses of 0.5, 1, 2 and S Gy. The control samples, and Irradiated cells, were maintained in culture medium for 24, 48 and 72 hours fellowing the Irradiation. The number of apoptotic cells after the in vitro X-irradiation was measured by flow cytometry after Incubation periods of 24, 48 and 72 hours. We also observed the apoptotic cells using a DNA fragmentation assay and electron microscopy. Results : The rate oi spontaneous apoptosis increased in relation to the time interval following irradiation (1.761 ${\pm}$0.161, 3.563${\pm}$0.554, 11.098${\pm}$2.849, at 24, 48, and 72 hours). The apoptotli cells also increased In the samples irradiated with 0.5, 1, 2 and 5 Gy, In a radiation dose and time interval after Irradiation manner, with the apoptosls being too great at 72 hours after Irradiation. The dose-response curves were characterized by an Initial steep Increase In the number of apoptotic cells for Irradiation doses below 2 Gy, with a flattening of the curves as the dose approached towards 5 Gy. Conclusion :The flow cytometric assay technique yielded adequate data, and required less than 1 mL of blood. The time and dose dependence of the radiation-induced apoptosis, was also shown. It is suggested that the adequate time Interval required for the evaluation of apoptosis would be 24 to 48 hours after blood sampling.

• Parasites, Hosts and Diseases
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• v.32 no.4
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• pp.249-258
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• 1994

Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

• The Korean Journal of Food And Nutrition
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• v.29 no.6
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• pp.998-1007
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• 2016

The objective of this study was to investigate the effects of a lettuce (Lactuca sativa L.) extract on the inflammation of human umbilical vein endothelial cell (HUVEC) and blood lipid improvement in hypercholesterolemic mice fed a high cholesterol diet. The lettuce extract (100% ethanol extract) inhibited the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in HUVEC treated with tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$). The lettuce extract suppressed the adhesion of THP-1 to TNF-${\alpha}$-treated HUVEC. The lettuce extract decreased the TNF-${\alpha}$-stimulated production of proinflammatory cytokine interleukin-6, interleukin-8 and chemokine monocyte chemotactic protein 1. In hypercholesterolemic mice, the lettuce extract reduced serum total cholesterol, triglyceride, and low-density lipoprotein-cholesterol level, while the lettuce extract elevated high-density lipoprotein-cholesterol level, resulting in the decrease of atherogenic index and cardiac risk factor level. These results suggested that lettuce extract can be an useful resource to show an anti-inflammatory effect and improve lipid metabolism.