• BMB Reports
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• v.36 no.4
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• pp.390-398
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• 2003

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.299-305
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula/blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine oviductal epithelial cell monolayers(POEC) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8∼16-cell and morula/blastocyst stage were 57.2, 48.2, 37.2 and 19.3% in Ham's F-10 with POEC, and 51.4, 41.2, 31.1, and 15.5% in TCM-HEPES with POEC, respectively. The above development rates to morula/blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TCM-HEPES with out POEC(P<0.05). The in vitro development rates to the morula/blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without POEC were 1.1∼1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with POEC in the two different media enhanced the development of fertilized eggs to morula/blastocyst stages in vitro. However, we didn't find out any difference for the in vitro development to morula/blastocyst stages between Ham's F-10 and TCM-HEPES media.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.241-248
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• 1996

This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17$\beta$ and heat inactivated fetal calf serum at 39$^{\circ}C$ under 5% $CO_2$ in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

• Journal of the Korean Home Economics Association
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• v.24 no.1
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• pp.43-51
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• 1986

In order to study antioxidative acton of garlic, alliin, scordinin, garlic oil, ethanol fraction and non-kaolin fraction which have been discovered from garlic until now were extracted and isolated, and each fraction was utilized as the experimental materials. Antioxidative action of each fraction was compared through the in vitro and in vivo experiments. Electron donatingability on $\alpha$, $\alpha$-diphenyl-$\beta$picrylhydra-$\chi$yl, the inhibitory effect of lipoperoxide formation by TBA and peroxide value were measured and analyzed. RESULTS : 1. When observed antioxidative ability by EDA value, ethanol fraction of garlic components showed the strongest reaction as 15.25. 2. In vitro experiment with TBA value, garlic oil, alliin and ethanol fracton showed distinctive effect on inhibitory effect of lipoperoxide formation. 3. Comparing with the inhibitory effect of lipoperoxide formation with TBA value in vivo, the ethanol fraction was the most effective in the blood or liver by intraperitoneal administration, whereas the ethanol fraction in the blood and non-kaolin fraction in the liver was most effective each other by orally administration. 4. In vitro experiment with peroxide value, garlic oil was distinctive effect on the inhivitory effect of lipoperoxide formation, which was a similar to the trend of TBA value in vitro. 5. Examining the induction time for the first period of lipoperoxide formation in vitro, garlix oil, ethanol fraction and alliin were effective, which was a similar to the trend of TBA value and peroxide value in vitro.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.355-362
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• 1997

This study was undertaken in an effort to product embryos through in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) after cryopreservation of immature and mature porcine oocytes. The experiments were conducted to investigate IVM rate of oocytes frozen with 3 different cryoprotectants and to examine IVF and IVC of frozen-thawed oocytes. The CEI(cumulus cells expansion index) after IVM of frozen-thawed immature oocytes was higher in oocytes frozen with PG＋PEG(propylene glycol plus polyethylene glycol) than those frozen with single cryoprotectant and this index was almost 90% of unfrozen oocyte's index(2.39 vs. 2.66). The IVF rate of all frozen oocytes was very low(68% of unfrozen oocytes) and the IVF rate of frozen immature oocytes was slightly higher than that of frozen mature oocytes(39.0% vs. 34.4%), but polyspermic penetration was higher in frozen immature oocytes(21.9% vs. 19.1%). The cleavage rate after IVF of frozen-thawed oocytes was 9.3% for frozen mature oocytes and 11.3% for frozen immature oocytes and this rate was significantly lower(P<0.05) than that of control(60.7%). The development to 8-cell stage was greatly lower in frozen mature oocytes than in frozen immature oocytes. The results indicate that the use of PG plus PEG as cryoprotectant may be very effective for vitrification of porcine oocytes and the frozen-thawed immature porcine oocytes can be used fro in vitro embryo production based on IVM, IVF and IVC system.

• Journal of Pharmaceutical Investigation
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• v.34 no.5
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• pp.393-399
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• 2004

For the efficient determination of activity against solid tumors, an in vitro tumor model that resembles the condition of in vivo solid tumors, is required. The purpose of this study was to establish a rapid culture method and viability assay for an in vitro 3-dimensional tumor model, multicellular spheroid (MCS). Among 12 human cancer cell lines, a few cell lines including DLD-1 (human colorectal carcinoma cells) formed fully compact MCS which was adequate for in vitro viability assay. DLD-1 MCS showed steady growth reaching $700\;{\mu}m$ diameter after 11 day culture. DLD-1 cells grown as MCS showed significant increase in $G_0/G_1$ phase compared to the monolayer cells (73.9% vs 45.7%), but necrotic regions or apoptotic cells were not observed. The cells cultured as MCS showed resistance to 5-FU (10.3 fold higher $IC_{50}$) compared to monolayers, however, tirapazamine (a hypotoxin) showed similar activity in both culture systems. In summary, MCS may be a valid in vitro model for activity screening of anticancer agents against human solid tumors and also exploitable for studying molecular markers of drug resistance in human solid tumors.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

• Environmental Analysis Health and Toxicology
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• v.12 no.3_4
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• pp.43-54
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• 1997

Species, doses and routes extrapolation can be sucessfully carried out by using a physiologically-based pharmacokinetic (PBPK) approach. And PBPK approach to assess risk of hazardous chemicals is reasonable whatever the exposure scenarios are happened. Both partitioning coefficients of chemical between tissue and blood and enzymatic metabolic rate constants are key parameters to build up the PBPK model. In this study, we tried to estimate in vitro metabolic rate constants using a special apparatus instead to measure the in vivo constants which are used to PBPK simulation since the in vitro tests are less expensive and more convenient than in vivo tests. For the purpose, we designed and tested the new system to measure continuously the headspace concentration of VOC. The newly designed system is composed with a diffusion chamber which generates gaseous substrate, a reaction vessel with a recirculating pump to establish a closed system, an autbmatic sampler from a gas phase, a gas chromatography to analyze the headspace. In addition, a cold water condenser is attached between the reaction vessel and pump to reduce the content of gaseous moisture which interferes with chemical analysis. To validate the newly developed methodology, in vitro metabolic rate constants of trichloroethylene (TCE) as a prototype VOC were estimated by simulating observed results with an ACSL program. The simulated results are consistent to those estimated by the other research groups. This finding suggests that our newly designed closed system may be a useful apparatus to estimate in vitro metabolic rate constants for VOC.

• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.528-528
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• 2003

The present study was undertaken to develop the in vitro sun protection factor(SPF) test method having good correlation with in vivo method using human. 8% homomentyl salicylate, P3 reference standard and commercially available sunscreen products were measured by the in vitro method using SPF 290S analyzer, and the SPFs were compared with the SPFs measured by in vivo test method. In vitro SPFs of 8% HMS and P3 reference standard were 4.59 $\pm$ 0.12 and 14.94 $\pm$ 0.83. There are good correspondence, correlation coefficients were 0.9506 and 0.9769 respectively, between the in vitro and in vivo SPFs for the sunscreen creams and lotions. Correlation coefficients of makeup base/liquid foundation, lotion labled with "shake before use" and compact powder were 0.8812, 0.8632 and 0.5984 respectively. The optimum mixture ratio of compact powder and cream base represents 1:0.8. These results suggest that the in vitro SPF test method will be able to be used as an alternative method for in vivo SPF in case of lotion and cream.