• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• Molecules and Cells
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• 제40권6호
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• pp.410-417
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• 2017

Estimation of postmortem interval (PMI) is a key issue in the field of forensic pathology. With the availability of quantitative analysis of RNA levels in postmortem tissues, several studies have assessed the postmortem degradation of constitutively expressed RNA species to estimate PMI. However, conventional RNA quantification as well as biochemical and physiological changes employed thus far have limitations related to standardization or normalization. The present study focuses on an interesting feature of the subdomains of certain RNA species, in which they are site-specifically cleaved during apoptotic cell death. We found that the D8 divergent domain of ribosomal RNA (rRNA) bearing cell death-related cleavage sites was rapidly removed during postmortem RNA degradation. In contrast to the fragile domain, the 5' terminal region of 28S rRNA was remarkably stable during the postmortem period. Importantly, the differences in the degradation rates between the two domains in mammalian 28S rRNA were highly proportional to increasing PMI with a significant linear correlation observed in mice as well as human autopsy tissues. In conclusion, we demonstrate that comparison of the degradation rates between domains of a single RNA species provides quantitative information on postmortem degradation states, which can be applied for the estimation of PMI.

• Clinical and Experimental Reproductive Medicine
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• 제39권3호
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• pp.114-117
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• 2012

Objective: It is well known that fresh blastocyst transfer results in better pregnancy outcomes with a smaller number of transferred embryos compared with cleavage stage embryo transfer. However, in terms of frozen-thawed blastocyst transfer, only a few studies are available. We aimed to evaluate clinical outcomes of frozen-thawed embryo transfer (FET) with blastocysts. Methods: Retrospective analysis of FET cycles with blastocysts (B-FET) between Jan 2007 and June 2009 was performed. Age-matched FET cycles with cleavage stage embryos (C-FET) during the same period were collected as controls. A total of 58 B-FET cycles were compared with 172 C-FET cycles and also compared with those of post-thaw extended culture blastocysts from frozen pronuclear stage embryos (22 cycles). Results: There was no difference in the patient characteristics of each group. The embryos' survival rates after thawing were comparable (>90%) and there was no difference in the implantation rate or clinical and ongoing pregnancy rate among the three groups. Conclusion: In FET, blastocyst transfers may not present better pregnancy outcomes than cleavage stage embryo transfers. A further large-scale prospective study is needed.

• 한국수정란이식학회지
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• 제15권2호
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• pp.129-136
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• 2000

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4$ vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 $\mu$1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제33권10호
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• pp.1579-1589
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• 2020

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

• 한국주조공학회지
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• 제25권1호
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• pp.16-22
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• 2005

This study aims to investigate the effects of the microstructures and nodule type on the fatigue characteristics of cast iron. Fatigue tests were carried out in tension-tension mode using a servo-hydraulic testing machine with load control mode operating at a frequency of 15 Hz. The tests were conducted at stress ratio R=Kmin/Kmax, of 0.1. Initial crack ${\Dalta}K$ values were highly performed with increase in tensile strength of DCI fatigue specimens. ${\Dalta}K_{th}$ region, fatigue crack propagation was primarily advanced through cell boundary and in periphery of near nodule. Fatigue crack propagation rate of D2 consisted with 2Phase(Ferrite+Pearlite) was slow due to crack closure enhanced by crack deflection and occurred crack branching. The generation of crack branch was occurred due to interaction of crack-nodule. At Threshold and Paris zone, the fractographs of the fatigue fracture surface for DCI show typical striations of a ductile fracture and isolated cleavage planes near graphite. The effect of microstructure on fatigue crack propagation of GC strongly depends on the type of flake. The generation of crack branch occurred due to interaction of crack-nodule. The fractographs of the fatigue fracture surface for GC show cleavage plane along the flake graphite.

• Asian-Australasian Journal of Animal Sciences
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• 제22권7호
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• pp.962-968
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• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.

• 한국주조공학회지
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• 제23권2호
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• pp.77-85
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• 2003

Some alloying elements such as Cr, B, Ti and Si were added individually or as a mixture to Fe-30 at.%Al alloys. The alloys were melted using an arc furnace and then heat-treated for homogenization at 1000$^{\circ}C$ for 7 days and followed by rolling at 1000$^{\circ}C$. The alloying elements on rolling characteristics were investigated by the microstructures and fracture mode before and after rolling. The microstructures before rolling showed that all of the alloys had equiaxed grains. On the other hand, the microstructures of rolling plane as well as its perpendicular plane became elongated after rolling. The alloys such as Fe-30Al, Fe-30Al-3Ti, Fe-30Al-0.5B, Fe-30Al-5Cr and Fe-30Al-3Ti-0.5B revealed better rolling behaviour from the point that intergranular and cleavage fractures were not fundamentally occurred. But the addition of 5Ti or 3Si to Fe-Al alloys had detrimental effects. The Ti-added alloy system such as Fe-30Al-5Ti, Fe-30Al-5Ti-5Cr, Fe-30Al-3Ti-5Cr and Fe-30Al-5Ti-0.5B were cracked through grain and showed cleavage fracture. The Si-added alloy system such as Fe-30Al-5Si, Fe-27Al-3Si and Fe-27Al-5Cr-3Si were cracked along the grain boundary and showed intergranular fracture. $DO_3{\leftrightarrow}B_2$ transition temperature of Fe-30at.%Al alloy was 520$^{\circ}C$, whereas the addition of 3Ti and 3Ti+0.5B comparably increased the temperature to 797 and 773$^{\circ}C$, respectively.

• 대한한방소아과학회지
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• 제20권1호
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• pp.257-272
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• 2006

Objective : In this study, the ability of Oriental medicine Samultanggamibang(SMTG) to induce apoptosis was investigated in B16F10 melanoma cells. Method : Tetrazolium-based colorimetric assay was performed for cytotoxicity test. Several new assays for the basis of biochemical events associated with apoptosis such as DNA fragmentation by a flow cytometry, caspase-3 activation and PARP cleavage by Western blotting should be carried out potentially useful for the basis of biochemical events associated with apoptosis such as a flow cytometry and caspase-3 activation. Results : (1) The number of B16F10 melanoma cells was less than 30 % after exposure to 1 mg/ml SMTG for 48 h. SMTG increased cytotoxicity of B16F10 melanoma cells in a dose- and time-dependent manner. (2) The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 21 % at 24 h and 25 % at 48 h after treatment with 1 mg/ml SMTG. (3) SMTG-induced apoptosis was accompained by the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymerase. (4) SMTG induces the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymearse and eventually leads to apoptosis through c-Jun NH2-terminal protein kinase (JNK)-dependent manner in B16F10 melanoma cells. Conclusion : SMTG had a strong cytotoxic effect of B16F10 melanoma cells.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.430-434
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• 2004

Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-$\beta$-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.