• Molecules and Cells
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• v.28 no.6
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• pp.529-536
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• 2009

Genome sequencing of the pig is being accelerated because of its importance as an evolutionary and biomedical model animal as well as a major livestock animal. However, information on expressed porcine genes is insufficient to allow annotation and use of the genomic information. A series of expressed sequence tags of 5' ends of five full-length enriched cDNA libraries (SUSFLECKs) were functionally characterized. SUSFLECKs were constructed from porcine abdominal fat, induced fat cells, loin muscle, liver, and pituitary gland, and were composed of non-normalized and normalized libraries. A total of 55,658 ESTs that were sequenced once from the 5′ ends of clones were produced and assembled into 17,684 unique sequences with 7,736 contigs and 9,948 singletons. In Gene Ontology analysis, two significant biological process leaf nodes were found: gluconeogenesis and translation elongation. In functional domain analysis based on the Pfam database, the beta transducin repeat domain of WD40 protein was the most frequently occurring domain. Twelve genes, including SLC25A6, EEF1G, EEF1A1, COX1, ACTA1, SLA, and ANXA2, were significantly more abundant in fat tissues than in loin muscle, liver, and pituitary gland in the SUSFLECKs. These characteristics of SUSFLECKs determined by EST analysis can provide important insight to discover the functional pathways in gene networks and to expand our understanding of energy metabolism in the pig.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.29-33
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• 2002

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured $3{\sim}13{\mu}M\;Ca^{2+}$ for 5 min., $5-8{\mu}g/ml$ cytoclacin for 6 hrs, 0.5~2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% $CO_2$, 95% air, $38^{\circ}C$. The cleavage rate after 48 hrs culture of oocytes treated with $3-13{\mu}M\;Ca^{2+}$, $5-8{\mu}g/ml$ cytoclacin and 0.5~2.0 mM DMAP for 5 min., 6 hrs and 3 hrs were 9.6%~20.0%, 0.0%~7.3% and 9.4%~21.8%, 0.0%~7.3% and 9.1%~21.8% and 0.0%~7.3%, respectively. When oocyte were treated with $10{\mu}M\;Ca^{2+}$, $10{\mu}g/ml$ cytoclacin and 2.0 mM DMAP the blastocyst formation rate was significantly higher than other group. The cleavage rate after 48 hrs culture of oocytes treated with $Ca^{2+}$ + cytoclacin, $Ca^{2+}$ + DMAP, cytoclacin + DMAP were 75.9%~93.5% and 9.7%~19.0%, respectively. When oocytes were treated with $Ca^{2+}$ followed by DMAP, the blastocyst formation rate was significantly higher than other group(p<0.05). When necleus transferred embryos co-cultured with bovine serum albumin(BSA), epithemal growth factor(EGF) and calf serum(CS), the developmental rate to blastocyst were higher than control group.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.121-126
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• 2007

The objective of this study was carried out to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as non-invasive marker to know the developmental competence in advance. The porcine oocytes matured for 48 h were examined the polar body extrusion. The examined oocytes were matured for additional $16{\sim}18h$ and activated with 7% ethanol and cultured in $5{\mu}g/ml$ cytochalasin B for 5 h for diploid formation. The treated oocytes were cultured and examined the cleavage after 48 h and continued culturing for 5 days. The oocytes of 21.9% were discarded in morphological selection and 32.1% oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated and then after 48 h the cleavage rates were examined. In morphologically selected oocytes, 15.8% oocytes were not cleaved and 52.6% oocytes were normally cleaved and 31.6% oocytes were hyper-cleaved over 8-cell stage. However in the first polar body extruded oocytes, 7.1% oocytes were not cleaved and 73.1% oocytes were normally cleaved and 19.8% oocytes were hyper-cleaved. The morphologically selected embryos that not cleavage-selected were developed in 16.7% up to blastocyst and the morphologically selected and cleavage-selected embryos were developed in 31.7%. The polar body extruded oocytes that were not carried out cleavage selection were developed in 39.0% and the polar body extruded and cleavage-selected embryos were developed 49.0%. The first cleavage timing was examined with 12 h interval after activation. In $0{\sim}12,\;12{\sim}24,\;24{\sim}36,\;and\;36{\sim}48h$ intervals, 4.1%, 68.6%, 19.1%, and 2.3% oocytes were cleaved and 5.9% oocytes were not cleaved until 48 after activation. The cleaved oocytes in each interval were cultured and developed upto blastocyst with 0, 39.1, 9.5, and 0%, respectively. This results suggests that polar body extruded and cleaved at $12{\sim}36h$ embryo has higher developmental potential than the others.

• Toxicological Research
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• v.12 no.2
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.215-221
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• 2002

Antioxidants(N-acetyl-1-cysteine, ebselen and glutathione) and growth factors(EGF, PDGF) were studied as a mean of increasing the development of porcine embryos produced by in vitro maturation (IVM) and in vitro fertilization(IVF). Porcine embryos developed to the 2~8 cell stage after IVF were cultured fer 6 to 7 days at 38.5$^{\circ}C$ in NCSU 23 medium containing antioxidants plus growth factors. Cell numbers of blastocysts were counted by fluorescence staining method. The developmental rate beyond morula stages in NCSU 23 containing NAC(1nm) or NAC(1nm) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 28.1, 32.3 and 35.3%, respectively. NAC plus PDGF group was slightly higher than control group(P>0.05). The developmental capacity in NCSU 23 containing ebselen(10 $\mu$M) or ebselen(10 $\mu$M) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 17.8, 36.9 and 40.3%, respectively Ebselen plus growth factor groups were significantly higher than control group(P<0.05). The developmental capacity in NCSU 23 containing glutathione(100 $\mu$M) or glutathione(100 $\mu$M) plus EGF (100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 24.1, 30.5 and 27.7%, respectively. There were not difference in all experimental groups(P>0.05). In all experimental groups, there was no significantly differences on the cell number of blastocysts, but ebselen plus growth factor groups were significantly higher than control group. These studies indicate that antioxidants plus growth factors can increase the proportion of embryos that developed beyond morulae stage.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.255-262
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• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.1-11
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• 2018

Cellular cyclic adenosine-3' 5'-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn't known. The purpose of this study is to improve the maturation of oocytes derived from follicles ${\leq}3mm$ in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the $1{\mu}M$ of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ${\leq}3mm$ in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.

• Food Science of Animal Resources
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• v.29 no.5
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• pp.579-589
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• 2009

This study was conducted to investigate the effects of heating, fiber type used in solid-phase microextraction (SPME, two phase vs three phase) and storage time on the volatile compounds of porcine M. longissimus dorsi (LD). Volatile compounds were measured using a gas chromatography and mass spectrometry (GC/MS) with a quadrupole mass analyzer. Among the volatile compounds identified, aldehydes (49.33%), alcohols (24.63%) and ketones (9.85%) were higher in pre-heated loins ($100^{\circ}C$/30 min), whereas, alcohols (34.33%), hydrocarbons (22.84%) and ketones (16.88%) were higher in non-heated loins. Heating of loins induced the formation of various volatile compounds such as aldehydes (hexanal) and alcohols. The total contents of hydrocarbons, alcohols, and carboxylic acids were higher in two phase fibers, whereas those of esters tended to be higher in three-phase fibers (p<0.05). Most volatile compounds increased (p<0.05) with increased storage time. Thus, the analysis of volatile compounds were affected by the fiber type, while heating and refrigerated storage of pork M. longissimus dorsi increased the volatile compounds derived from lipid oxidation and amino acid catabolism, respectively.

• Korean Journal of Veterinary Research
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• v.36 no.2
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• pp.395-403
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• 1996

Monoclonal antibodies(MAbs) against field isolates of the bovine rotavirus A strain(G6), V strain(G10) and reference I-801 strain(G8) were produced and characterized. Six MAbs(4C2, 4D9, 5E1, 5E7, 5D5, 3E4) against A strain had neutralizing activity and reacted only with the G6 bovine rotaviruses determined by fluorescence focus neutralization (FFN) test. Otherwise, five neutralizing MAbs(1G2, 2G6, 5E2, 5E12, 5H7) against I-801 strain neutralized the G6 and G8 bovine rotaviruses. Five non-neutralizing MAbs(5F12, 7F12, 5E11, 2A11, 2B12) were VP6-specific and cross-reacted with all bovine and porcine rotaviruses examined by fluorescence antibody(FA) test. None of the MAbs reacted with bovie viral diarrhea virus(BVDV) and bovine coronavirus(BCV) determined by FA and FFN test.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.3
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• pp.367-375
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• 1998

The physicochemical properties of the $\alpha$-amylase inhibitors from black bean and naked barley is Korea were investigated. Preincubation time for maximum inhibition was 30min and no activity change was seen after that time. Optimum pH of the $\alpha$-amylase inhibitors from the black bean and naked barley was pH 7.0 and the inhibitory activities were stable in the range of pH 6.0~8.0 in both phosphate and Tris-HCI buffer solutions. Both inhibitors maintained more than 50% of activity after incubation for 17 min at 7$0^{\circ}C$. The inhibitors from the black bean and naked barley maintained more than 50% of activities after treatment for 40 min and 30 min with pepsin, and 30 min and 50 min with trypsin, respectively. Both inhibitors functioned via a noncompetitive mechanism and were active against porcine pancreatic and human salivary $\alpha$-amylases. The activities of both inhibitors were linear for the ionic stength ranging from 0 to 0.9. The addition of 70 mM maltose to the reaction mixture caused a maximum increase in the relative activities of both inhibitors, but it did not affect the dissociation of the EI complex. The activities of both inhibitors were significantly enhanced by adding 1mM of K+ or Mg2+.