• Journal of fish pathology
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• v.22 no.3
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• pp.335-342
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• 2009

The stability of immunoglobulin M (IgM) on different serum storage conditions and specific antibody response were tested using the serum collected from sevenband grouper Epinephelus septemfasciatus by enzyme-linked immunosorbent assay (ELISA). To test the effect of storage temperature and duration, sevenband grouper antiserum against bovine serum albumin (BSA) was stored at -80, -20 or 4${^{\circ}C}$ for 1, 34, 61 or 119 days. In addition, to test the effect of repeated freeze-thawing condition, the anti-BSA fish serum was frozen at -20 and -80${^{\circ}C}$ and then thawn and frozen for 1, 5 or 10 times repeatedly. Consequently, no significant difference was found in ELISA optical density (O.D.) values of sera for the above mentioned storage conditions: different temperatures (-80, -20 and 4${^{\circ}C}$), durations of storage (1, 34, 61 and 119 days), and repeated thaw-freeze cycles (1, 5, and 10 times), indicating that IgMs of test fish were stable. The specific antibody response of sevenband grouper was observed after BSA-immunization of the test fish reared at 20 ${^{\circ}C}$ or 25${^{\circ}C}$. At the rearing temperature of 20${^{\circ}C}$, the specific antibody against BSA first appeared at 14 days and maximum antibody titer was observed between 21 and 28 days, while at the rearing temperature of 25 ${^{\circ}C}$, specific antibody appeared at 7 days and maximum antibody titer was observed between 14 and 21 days. In conclusion, the rearing temperature at 25${^{\circ}C}$ gave a faster and higher specific antibody response than at 20${^{\circ}C}$ and the specific antibody response maintained for approximately 2 months at 20℃ and 25${^{\circ}C}$.

• Food Science of Animal Resources
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• v.29 no.2
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• pp.151-156
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• 2009

The prevalence of food allergies was investigated using questionnaires with 300 subjects whose ages ranged from 19 to 24 years old and the causative food allergens was analyzed using immunological analysis with serum of the subjects who answered that they have/had food allergy. The questionnaire showed that 11.33% of subjects have/had experience of food hypersensitivity, where the main causative foods were fish, beef, chicken, milk, egg, and pork in order. The meat allergy shared 4.65% (2.33% for beef, 1.66% for chicken, 0.66% for pork) in the prevalence of food allergies. The causative beef allergens were investigated with the serum of 6 subjects who have had beef allergy. Western blots were carried out with the serum of P6 subject who showed a positive reaction to beef extract in ELISA. The two specific bands were detected in beef extract on the PVDF membrane, and no band was detected in extracts of pork and chicken. A calculation of the distance of migration by SDS-PAGE enabled the molecular masses of the two bands to be estimated as 67kDa and 31kDa, respectively. The 67kDa was revealed as bovine serum albumin (BSA) which is one of the important beef allergens as reported previously though an analysis of the N-terminal amino acid sequence. However we could not identify the sequence of 31kDa, probably because they comprised several subunits and were modified proteins such as glycoprotein that were unlikely to be easily degraded by the Edman method. The 31kDa band were dyed with the PAS (periodic acid-schiff reagent), suggesting that it might be a glycoprotein. These results suggested that the 31kDa might be considered as a novel potential beef allergen which is not reported previously, although further studies are needed.

• Korean Chemical Engineering Research
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• v.43 no.1
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• pp.103-109
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• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.157-162
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• 2006

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs (cumulus oocytes complexes) in vitro. Attempts had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of FGF (fibroblast growth factor) on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in HPM 199 (Inst. of Functional peptide, Japan) containing 0.1, 1 and 10 ng/ml FGF for 24 hr, maturation rates to metaphase II ($70.0{\sim}75.0%$) were significantly higher (p<0.05) than that of control group (0 ng/ml FGF, 37.5%). When matured COCs with FGF were cultured in maturation medium after in vitro fertilization, developmental rates to blastocysts were 9.5, 0 and 2.9%, respectively, compared to 25.0% of the control group (p<0.05). When the matured COCs with FGF were cultured in HPM 199 (IFP971, Inst. of Functional peptide, Japan) containing 10% FBS, 0.8% BSA or 0.1% PVA (polyvinyl alcohol), the blastocyst formation rates were 12.4, 12.8 and 8.5%, respectively, while the rates of matured COCs with FGF and cultured with IVMD and IVD (Inst. of Functional peptide, Japan) without serum were 38.4% and 34.8%, respectively (p<0.05). These results suggested that FGF is available for in vitro maturation of bovine COCs and is not suitable for in vitro development, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine embryo development.

• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Journal of Life Science
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• v.25 no.4
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• pp.416-424
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• 2015

Pinosylvin is a stilbenoid found in the Pinus species. Pinosylvin at ~pM to ~nM concentrations induces cell proliferation, cell migration and anti-inflammatory activity in endothelial cells. However, it was recently reported that pinosylvin at high concentrations (50 to 100 μM) induces cell death in bovine aortic endothelial cells. In this study, we conducted a series of experiments to discover how pinosylvin at a high concentration (50 μM) induces endothelial cell death. Pinosylvin at the high concentration was shown to induce endothelial cell apoptosis through enhancing caspase-3 activity, flip-flop of phosphatidyl serine, and nuclear fragmentation. We found that pinosylvin at the high concentration additively increased caspase-3 activity enhanced by serum-starvation or treatment with 100 μM etoposide. We also determined that pinosylvin at the high concentration promoted activations of c-Jun N-terminal kinase (JNK) and endothelial nitric oxide synthetase (eNOS). We further ran a series of experiments to find out which signaling molecule plays a critical role in the pinosylvin-induced apoptosis. We finally found that SP-600125, a JNK inhibitor, had an inhibitory effect on the pinosylvin-induced endothelial cell death, but L-NAME, an eNOS inhibitor, had no effect. These data indicate that JNK is involved in the pinosylvin-induced apoptosis. Collectively, pinosylvin at high doses induces cell apoptosis via JNK activation.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.247-250
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• 2008

The $CHCl_3$, EtOAc, and n-BuOH fractions showed a marked inhibition of 5% fatal bovine serum (FBS)-induced cell proliferation. The $IC_{50}$ values of the chloroform fractions from leaf, stem, and root as well as the n-BuOH and EtOAc fraction from root on cell proliferation were $1.2{\pm}0.4$, $17.2{\pm}6.4$, $81.8{\pm}33.2$, $40.8{\pm}8.0$, and $237.1{\pm}85.6\;{\mu}g/mL$, respectively. On the other hand, the EtOAc fractions, and the $CHCl_3$ fraction significantly inhibited collagen-, arachidonic acid-, U46619-, and thrombin-induced platelet aggregations. The $IC_{50}$ values of EtOAc fraction from leaf, and the $CHCl_3$ and EtOAc fraction from stem were $214.1{\pm}12.2$, $134.3{\pm}2.5$, and $42.6{\pm}7.0\;{\mu}g/mL$ with collagen, $312.4{\pm}7.5$, $158.9{\pm}1.7$, and $82.2{\pm}2.7\;{\mu}g/mL$ with arachidonic acid, $31.1{\pm}2.4$, $48.7{\pm}0.3$, and $29.7{\pm}1.1\;{\mu}g/mL$ with U46619, and $36.7{\pm}2.4$, $69.1{\pm}11.3$, and $34.2{\pm}0.1\;{\mu}g/mL$ with thrombin, respectively. Taken together, these data provide new evidence that fractions from Allium victorialis var. platyphyllum (AVP) are able to inhibit vascular smooth muscle cell (VSMC) proliferation and platelet aggregation, which may be a novel resource for the development of anti-atherothrombotic agents.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.35-41
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• 2014

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot ($20mg{\times}6/300IU$) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ${\pm}$ SEM) observed which developed to larger than 2 mm in diameter were $1.6{\pm}2.5$, $4.3{\pm}3.1$, $5.5{\pm}4.2$, $6.6{\pm}2.1$ and $8.8{\pm}7.8$, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + $10{\mu}g/ml$ FSH + $1{\mu}g/ml$ $17{\beta}$-estradiol for 27 h at $39^{\circ}C$ in 5% $CO_2$ in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6-DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.