• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.139-147
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• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• 한국산업보건학회지
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• 제17권3호
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• pp.235-244
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• 2007

By comparing the proteins from the workers exposed to styrene with the ones from controls, it may be possible to identify proteins that play a role in the occurrence and progress of occupational disease and thus to study the molecular mechanisms of occupational disease. In order to find the biomarkers for assessing the styrene effects early, before clinical symptoms develop and to understand the mechanisms of adverse health effects, we surveyed 134 employees, among whom 52 workers(30 male and 22 female) were chronically exposed to styrene in 10 glass-reinforced plastic boat manufacturing factories in Korea and 82 controls had never been occupationally exposed to hazardous chemicals including styrene. The age and drinking habits and serum biochemistry such as total protein, BUN and serum creatinine in both groups were significantly different. Exposed workers were divided into three groups according to exposure levels of styrene(G1, below 1/2 TLV; G2, 1/2 TLV to TLV; G3, above TLV). The mean concentration of airborne styrene in G1 group was $10.93{\pm}11.33ppm$, and those of urinary mandelic acid(MA) and phenylglyoxylic acid(PGA) were $0.17{\pm}0.21$ and $0.13{\pm}0.11g/g$ creatinine, respectively. The mean concentration of airborne styrene in G2 and G3 groups were $47.54{\pm}22.43$ and $65.33{\pm}33.47ppm$, respectively, and levels of urinary metabolites such as MA and PGA increased considerably as expected with the increase in exposure level of styrene. The airborne styrene concentration were significantly correlated to the urinary concentration of MA(r=0.784, p=0.000) and PGA(r=0.626, p<0.001). In the 2D electrophoresis, the concentration of five proteins including complement C3 precursor, alpha-1-antitrypsin(AAT), vitamin D binding protein precursor(DBP), alpha-1-B-glycoprotein(A1BG) and inter alpha trypsin inhibitor(ITI) heavy chain-related protein were significantly altered in workers exposed to styrene compared with controls. While expression of complement C3 precursor and AAT increased by exposure to styrene, expression of DBP, A1BG and ITI heavy chain-related protein decreased. These results suggest that the exposure of styrene might affects levels of plasma proteinase, carriers of endogenous substances and immune system. In particular, increasing of AAT with the increase in exposure level of styrene can explain the tissue damage and inflammation by the imbalance of proteinase/antiproteinase and decrease of DBP, A1BG and ITI heavy chain-related protein in workers exposed to styrene is associated with dysfunction and/or declination in immune system and signal transduction

• 대한한방내과학회지
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• 제29권3호
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• pp.554-566
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• 2008

Objectives : Modified Gamgil-tang is a prescription commonly used for respiratory diseases. This thesis was carried out to check the treatment effects and diversity of drug formulation by comparing extraction method of ethanol and water of modified Gamgil-tang. Methods : All experiments were carried out with water and 50% ethanol extraction for comparison. In vivo experiment, hyaluronidase inhibitory effects and trypsin inhibitory effects were tested to measure the anti-inflammatory effects activity. Scavenging effects of DPPH free radical, xanthine oxidase inhibitory effects and inhibition on TBA-RS formation were experimented to measure anti-oxidative effects. With the in vivo experiment, ICR group mice and SD group rats were used as experimental animals. An anti-inflammatory effects experiment were carried out to measure the action on carrageenin-induced hind paw edema: analgesic effects were measured using writhing syndrome induced by 0.7% acetic acid in mice: antipyretic effect was measured using endotoxin, and inhibitory effects of increase vascular permeability induced by 0.5% histamine were measured. Results : For extraction of glycyrrhizin contents, ethanol extract was extracted 2 times of that of water extract. Anti-inflammatory effects showed high in ethanol extract. Anti-oxidative effects measured high in ethanol extract. No significant result was found in inhibition on TBA-RS formation. Analgesic effects were found to be similar in water and ethanol extract. Antipyretic effects were found to be stronger in water extract. Inhibitory effects of increase vascular permeability induced by 0.5% histamine showed stronger in ethanol extract. Conclusion : By measuring anti-inflammatory effects, analgesic effects, antipyretic effects, anti-oxidative effects, and histamine permeation inhibition effects both in water extract and ethanol extract after adding agents such as Mentha Herba, Gardenias Fructus, and propolis to existing Gamgil-Tang, ethanol extract was found to be more effective in anti-inflammatory effects, analgesic effects, anti-oxidative effects, and histamine permeation inhibition effects. The converse was found for antipyretic effect.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.534-541
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• 2009

The aim of this study was to evaluate the effects of gamma irradiation (${\gamma}$-irradiation) at doses of 15, 30 and 45 kGy on chemical composition, anti-nutritional factors, ruminal dry matter (DM) and crude protein (CP) degradibility, in vitro CP digestibility and to monitor the fate of true proteins of full-fat soybean (SB) in the rumen. Nylon bags of untreated or ${\gamma}$-irradiated SB were suspended in the rumens of three ruminally-fistulated bulls for up to 48 h and resulting data were fitted to a nonlinear degradation model to calculate degradation parameters of DM and CP. Proteins of untreated and treated SB bag residues were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Digestibility of rumen undegraded CP was estimated using the three-step in vitro procedure. The chemical composition of raw and irradiated soybeans was similar. Results showed that phytic acid in ${\gamma}$-irradiated SB at dose of 30 kGy was eliminated completely. The trypsin inhibitor activity of 15, 30 and 45 kGy ${\gamma}$-irradiated SB was decreased (p<0.01) by 18.4, 55.5 and 63.5%, respectively. From in sacco results, ${\gamma}$-irradiation decreased (p<0.05) the washout fractions of DM and CP at doses of 30 and 45 kGy, but increased (p<0.05) the potentially degradable fractions. Gamma irradiation at doses of 15, 30 and 45 kGy decreased (p<0.05) effective degradability of CP at a rumen outflow rate of 0.05 $h^{-1}$ by 4.4, 14.4 and 26.5%, respectively. On the contrary, digestibility of ruminally undegraded CP of irradiated SB at doses of 30 and 45 kGy was improved (p<0.05) by 12 and 28%, respectively. Electrophoretic analysis of untreated soybean proteins incubated in the rumen revealed that ${\beta}$-conglycinin subunits had disappeared at 2 h of incubation time, whereas the subunits of glycinin were more resistant to degradation until 16 h of incubation. From the SDS-PAGE patterns, acidic subunits of 15, 30 and 45 kGy ${\gamma}$-irradiated SB disappeared after 8, 8 and 16 h of incubation, respectively, while the basic subunits of glycinin were not degraded completely until 24, 48 and 48 h of incubation, respectively. It was concluded that ${\gamma}$-irradiated soybean proteins at doses higher than 15 kGy could be effectively protected from ruminal degradation.

• Asian-Australasian Journal of Animal Sciences
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• 제23권12호
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• pp.1614-1625
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• 2010

Two experiments were conducted to evaluate the use of fish silage prepared from Indian oil sardines, Sardinella longiceps, as partial replacement of soybean meal as a sole source of protein for growing broiler chickens. The main objective of Experiment 1, an ileal digestibility assay, was to assess the nutritional value of fish silage compared with soybean meal for feeding broiler chickens. The two test ingredients, soybean meal and dried fish silage, were incorporated into semi-synthetic diets, as the only component containing protein. The ileal digestibility coefficients of amino acids of fish silage were considerably higher than those of soybean meal (p<0.001). The lower digestibility of amino acids from soybean meal was related to the presence of anti-nutritional factors such as trypsin inhibitors. Fish silage had higher levels of sulphur-containing amino acids than soybean meal. The objective of Experiment 2, a growth study, was to evaluate the effect of feeding fish silage on performance and meat quality characteristics of broiler chickens raised under closed and open-sided housing systems. Four diets containing various levels of fish silage (0, 10, 20 and 30%) were evaluated. Daily feed intake, body weight gain and feed conversion ratio were measured. At the end of Experiment 2, 96 birds were randomly selected and slaughtered to evaluate meat quality characteristics. Housing type had significant effects on feed intake and body weight gain (p<0.01). Birds in the open-sided house consumed 4.7% less amount of feed and gained 10.6% less than their counterparts in a closed house. Birds in both houses fed diets containing 10 and 20% fish silage gained more than birds fed 30% fish silage. The current study produced evidence that fish silage can replace up to 20% of soybean meal in broiler diets without affecting either growth performance or the sensory quality of broiler meat.

• Interdisciplinary Bio Central
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• 제1권3호
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• pp.9.1-9.6
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• 2009

Introduction: In the mass spectrometry-based proteomics, biological samples are analyzed to identify proteins by mass spectrometer and database search. Database search is the process to select the best matches to the experimental mass spectra among the amino acid sequence database and we identify the protein as the matched sequence. The match score is defined to find the matches from the database and declare the highest scored hit as the most probable protein. According to the score definition, search result varies. In this study, the difference among search results of different search engines or different databases was investigated, in order to suggest a better way to identify more proteins with higher reliability. Materials and Methods: The protein extract of human mesenchymal stem cell was separated by several bands by one-dimensional electrophorysis. One-dimensional gel was excised one by one, digested by trypsin and analyzed by a mass spectrometer, FT LTQ. The tandem mass (MS/MS) spectra of peptide ions were applied to the database search of X!Tandem, Mascot and Sequest search engines with IPI human database and SwissProt database. The search result was filtered by several threshold probability values of the Trans-Proteomic Pipeline (TPP) of the Institute for Systems Biology. The analysis of the output which was generated from TPP was performed. Results and Discussion: For each MS/MS spectrum, the peptide sequences which were identified from different conditions such as search engines, threshold probability, and sequence database were compared. The main difference of peptide identification at high threshold probability was caused by not the difference of sequence database but the difference of the score. As the threshold probability decreases, the missed peptides appeared. Conversely, in the extremely high threshold level, we missed many true assignments. Conclusion and Prospects: The different identification result of the search engines was mainly caused by the different scoring algorithms. Usually in proteomics high-scored peptides are selected and low-scored peptides are discarded. Many of them are true negatives. By integrating the search results from different parameter and different search engines, the protein identification process can be improved.

• Asian-Australasian Journal of Animal Sciences
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• 제31권10호
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• pp.1660-1669
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• 2018

Objective: This study was conducted to evaluate the effect of probiotics (Bacillus subtilis and Enterococcus faecium) and xylo-oligosaccharide (XOS) supplementation on growth performance, nutrient digestibility, serum profiles, intestinal health, fecal microbiota and noxious gas emission in weanling pigs. Methods: A total of 240 weanling pigs ([Yorkshire${\times}$Landrace]${\times}$Duroc) with an average body weight (BW) of $6.3{\pm}0.15kg$ were used in this 28-day trial. Pigs were randomly allocated in 1 of the following 4 dietary treatments in a $2{\times}2$ factorial arrangement with 2 levels of probiotics (0 and 500 mg/kg probiotics) and XOS (0 and 200 mg/kg XOS) based on the BW and sex. Results: Administration of probiotics or XOS improved average daily gain (p<0.05) during 0 to 14 d and the overall period, while pigs that were treated with XOS had a greater average daily gain and feed efficiency (p<0.05) compared with unsupplemented treatments throughout 15 to 28 d and the whole experiment. Either probiotics or XOS treatments increased the apparent total tract digestibility of nutrients (p<0.05) during 0 to 14 d. No effects on serum profiles were observed among treatments. The XOS increased villus height: crypt depth ratio in jejunum (p<0.05). The supplementation of probiotics (500 mg/kg) or XOS (200 mg/kg) alone improved the apparent total tract digestibility of dry matter, nitrogen and gross energy on d 14, the activity of trypsin and decreased fecal NH3 concentration (p<0.05). Administration of XOS decreased fecal Escherichia coli counts (p<0.05), while increased lactobacilli (p<0.05) on d 14. There was no interaction between dietary supplementation of probiotics and XOS. Conclusion: Inclusion of XOS at 200 mg/kg or probiotics (Bacillus subtilis and Enterococcus faecium) at 500 mg/kg in diets containing no antibiotics significantly improved the growth performance of weanling pigs. Once XOS is supplemented, further providing of probiotics is not needed since it exerts little additional effects.

• 한국수산과학회지
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• 제51권2호
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• pp.127-134
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• 2018

Arteriosclerosis is the major cause of coronary artery and cerebrovascular disease, which are leading causes of death. Pro-inflammatory cytokines induce injury to vascular endothelial cells by increasing cell adhesion molecules, leading to vascular inflammation, a major risk factor for the development of arteriosclerosis. In the current study, we investigated the inhibitory effect of enzymatic hydrolysate from Japanese mud shrimp Upogebia major on the inflammation of tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$)-stimulated human umbilical vein endothelial cells (HUVECs). We first evaluated the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of eight U. major enzymatic hydrolysates: alcalase, papain, ${\alpha}$-chymotrypsin (${\alpha}-Chy$), trypsin, pepsin, neutrase, protamex and flavourzyme. Of these, ${\alpha}-Chy$ exhibited potent antioxidant and ACE inhibitory activities. The ${\alpha}-Chy$ hydrolysate was fractionated by two ultrafiltration membranes of 3 and 10 kDa. The ${\alpha}-Chy$ hydrolysate of U. major and its molecular weight cut-off fractions resulted in a significant reduction in NO production and a decrease in cell adhesion molecules [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] and pro-inflammatory cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)] in $TNF-{\alpha}$-stimulated HUVECs. These results suggest that enzymatic hydrolysate from U. major can be used in the control and prevention of vascular inflammation and arteriosclerosis.

• Asian-Australasian Journal of Animal Sciences
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• 제14권11호
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• pp.1598-1604
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• 2001

Eight $12.4{\pm}0.6kg$ initial body weight crossbred barrows were used to determine the effect of soybean galactooligosaccharides on nutrient and energy digestibility, and digesta transit time. Four dietary treatments were utilized in this trial. Treatment one was a corn-soybean meal based diet (SBM) containing raffinose and stachyose at the levels of 0.16% and 0.75%, respectively. Treatment two (control) was a corn-HP300 (soybean concentrate protein) diet. In treatments three and four, 1.1% and 2.2% commercial stachyose was added to the control diet to provide total dietary stachyose at the levels of 1% and 2%, respectively. The soybean galactooligosaccharides (raffinose + stachyose) level in treatment one was slightly lower compared to that in treatment three. Three collection periods were run with two pigs for each treatment/period. There was a 4 d adjustment period followed by a 3 d collection period. The results showed that the nitrogen retention (86.79%) of pigs fed treatment two diet was higher than that of pigs fed treatment one by 5.2% (p<0.05). The nitrogen retention of treatment three was intermediate 83.09%. The apparent fecal digestibility of all amino acids in treatment two was numerically highest, followed by treatments three and four. However, there were no significant difference among groups (p>0.05). The dry matter (DM), organic matter (OM), crude protein (CP), and crude fiber (CF) digestibility numerically decreased as the soybean galactooligosaccharides level increased, but were not significantly different (p>0.05). Chromium content in feces (from the inclusion of 0.3% chromic oxide in the diets) differed among treatments (p<0.05) at 15 h, 18 h, and 21 h after eating. This showed that the digesta transit time was differed significantly among treatments. Treatment four was the shortest, followed by treatment three, SBM and control. The results demonstrated that in the absence of antinutritional factors and soybean antigen protein, inclusion of 1% and 2% stachyose in corn-HP300 diet has no significant effect on the digestibility of DM, OM, CP, CF and amino acids. When the soybean galactooligosaccharide level in diet one and diet three were adjusted to be almost the same, antinutritional factors such as trypsin inhibitor and soybean antigen protein could decrease the nutrient digestibility and nitrogen retention rate of diet. High levels of soybean galactooligosaccharides shortened the digesta transit time in the intestinal tract. This trial suggested that the total level of soybean galactooligosaccharides (stachyose+raffinose) in the weanling piglet diet is better not to exceed 1% when common soybean meal is used as main protein source.