• Journal of Life Science
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• v.18 no.7
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• pp.952-957
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• 2008

We evaluated the effects of various factors (e.g., serum, inhibitors of protein synthesis, and cytoskeleton and protein kinases) on early PMA-induced THP-1 cell adhesion using an adhesion assay with Sulforhodamine B (SRB) staining, which was used to assess the proliferation of the attached cells. THP-1 cell adhesion to a plastic substrate was detected 1 hr after exposure to Phorbol 12-Myristate 13-Acetate (PMA) and peaked after 18 hr. At concentrations > 25 nM PMA, the level of adhesion did not change. Based on our preliminary results, we used 25 nM PMA and 5 hr of culture as standard assay conditions. Early PMA-induced cell adhesion was not affected by the presence of serum or PD 98059 in the culture medium, but was affected by the addition of PKC inhibitors and cycloheximide. In the presence of actin inhibitor with PMA, the cell adhesion increased when comparing with PMA treatment only. Thus, early PMA-induced adhesion of THP-1 cells does not require serum in the culture medium, MAP-kinase activation, or actin polymerization, but does require de novo protein synthesis and PKC activation. Our SRB-based cell adhesion assay may be used to screen other PKC inhibitors.

• Journal of the Korean Society of Tobacco Science
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• v.26 no.2 s.52
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• pp.152-158
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• 2004

Among short-term in vitro genotoxicity assays, micronucleus assays are rapid, inexpensive, and less labor-intensive system. We have undertaken a comparative study of sensitivity of cigarette smoke condensate(CSC) by general micronucleus(MN) assay and cytokinesis-block micronucleus(CBMN) assay. In this study, V79 Chinese hamster cells were employed to evaluate and compare the genotoxicity of CSC of Kentucky Reference Cigarette 2R4F by 2 kinds of in vitro MN assay methods. To determine the optimum concentration of cytochalasin B(CYB) to obtain the maximal number of binucleated cells for CBMN assay, triplicate cultures of growing cells were treated with CYB for 15 h. CYB treatments caused a concentration-dependent increase in cytotoxicity($1\~4{\mu}g/mL$) and proportion($0.25\~1\;{\mu}g/mL$) of binucleated cells. These data suggested that 1 ug/mL of CYB is as an optimum dose for CBMN assay in binucleated V79 cells. Short treatment(4 h) of CSC induced a micronucleated cells with a concentration-dependent response in the presence or absence of CYB, but CSC-induced MNs were weakened when S9 was present. Long treatments(19 h) of CSC also induced a significant increase MN formation with a concentration-dependent response. At a concentration of 75 ${mu}g/mL$, the MN cell frequencies of general MN assay and CBMN assay were $6.5\%\;and\;11.7\%$, respectively. Linear regression analysis revealed a good correlation in CBMN assay between a concentration of CSC and MN cell frequency. All these data indicated that CBMN assay is more sensitive to the induction CSC-induced MN than general MN assay.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.313-317
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• 2011

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM $SrCl_2$ in Ca-free CZB medium in the presence of 5 II ${\mu}$g/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6~7 week-old mice(53.3%) than in oocytes from 8~9(80.9%) and 10~11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium(51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6~7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.247-253
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• 2000

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 $\mu\textrm{g}$/mL), and higher rate of encleation was obtained at 7.5 and 15 $\mu\textrm{g}$/mL than at $\mu\textrm{g}$/mL. In Experiment 2, oocytes enucleated in 7.5 $\mu\textrm{g}$/mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 $mutextrm{s}$) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 $mutextrm{s}$ or 90 $mutextrm{s}$, but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM ＋ 10% FBS in 5% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.125-133
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• 2006

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ${\sim}45$ day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, $30{\mu}sec$) and cultured with $7.5{\mu}g/ml$ cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at $39^{\circ}C,\;5%\;CO_2,\;5%\l;O_2$ in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT ($79.3{\pm}8.5\;and\;25.5{\pm}6.1,\;and\;85.0{\pm}6.4\;and\;38.6{\pm}5.5$, respectively)than NT counterparts ($65.1{\pm}5.2\;and\;15.6{\pm}3.0$, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT ($34.7{\pm}5.8\;and\;38.1{\pm}4.1$) than NT embryos ($24.8{\pm}3.2$). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.

• Biomedical Science Letters
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• v.20 no.4
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• pp.250-255
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• 2014

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit ($RpoB_{127-135}$) could be induced in TB patients expressing HLA-$A^*0201$ subtype. In order to examine whether $RpoB_{127-135}$ specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for $RpoB_{127-135}$ peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-$A^*0201$ subjects by in vitro immunization technique. In this study, we observed $RpoB_{127-135}$ specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. $RpoB_{127-135}$ specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-$A^*0206$ subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of $RpoB_{127-135}$ peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, $RpoB_{127-135}$, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, $RpoB_{127-135}$ specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the $RpoB_{127-135}$ peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.76-76
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• 2002

본 실험은 Piezo-미세조작기(PrimeTech Ltd., Japan)를 사용하여 마우스 핵이식 후 재구축배를 CZB와 KSOM 두가지 배양액을 사용하여 체외배양성적을 비교 검토하였다. MII의 미수정란은 성숙한 4~5주령 B6D2Fl에 hCG 주사 후 14시간째에 과적 방법을 통해 난관의 팽대부로 부터 회수하였고, metaphase II chromosome-spindle complex와 최소량의 세포질을 내경이 10$\mu\textrm{m}$인 피펫으로 흡입하여 탈핵하였다. 핵이식에 사용된 난구세포(8-l0$\mu\textrm{m}$)는 3시간동안 12% PVP 에처리 하여 piezo-미세조작기를 이용하여 세포질에 세포의 핵을 직접 미세주입 하였다. 핵이식 후 생존한 재구축배는 2시간동안 배양한 후 10mM SrC1$_2$와 5$\mu\textrm{g}$/$m\ell$의 cytochalasin B가 첨가된 $Ca^{2＋}$-free CZB에서 6시간 활성화 처리하였다, 활성화 처리 후 위전핵이 관찰된 재구축란을 CZB 와 KSOM 배지에서 배양하면서 발달률을 비교하였고, 상실배 및 배반포배로 발달한 재구축배를 day 3 대리모에 이식하였다. 표 1에서 보는 바와 같이 재구축배의 2-cell로의 발달률에 있어서 KSOM이 CZB에 비하여 유의적으로 높게 나타났으며(P<0.05), 또한 4-cell과 상실배/배반 포배로의 발달률에 있어서도 KSOM이 CZB에 비하여 유의적으로 높은 발달률을 나타내었다(P<0.01). 또한 KSOM 배지에서 배양된 상실배/배반포배를 대리모에 이식한 경우에 11.5 d.p.c에 생존한 태아가 관찰되었다. 이상의 결과로 핵이식 재구축배의 활성화 처리 후의 발생에는 KSOM 배지가 CZB 배지에 비하여 유효함을 확인 할 수 있었다.그와 같은 배양 기술을 이용하여 외래유전자를 도입한 일련의 결과에 관하여 보고 하고자한다., 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.로 우점하였다. 여름철 식물플랑크톤 대발생에 영향은 수온과 직산염이 중요하였으나, 부유물질 크게 기여하지 못하였다.애를 확인하고 지도 관점을 파악하는 것을 포함한다. 그러나 본 논문은 역사발생적 수학 학습-지도 원리의 실제적인 적용에 관하여는 기초적인 연구에 지나지 않기 때문에, 역사발생적 원리를 학교수학에 실제적으로 적용하기 위해서는 각각의 내용에 대한 철저한 역사적 분석을 바탕으로 하는 후속 연구가 필요하다./TEX>구성교육${\lrcorner}$이 조선총독부의 관리하에서 실행되었다는 것을, 당시의 사범학교를 중심으로 한 교육조직을 기술한 문헌에 의해 규명시켰다.nd of letter design which represents -natural objects and was popular at the time of Yukjo Dynasty, and there are some documents of that period left both in Japan and Korea. "Hyojedo" in Korea is supposed to have been influenced by the letter design. Asite- is also considered to have been "Japanese Letter Jobcheso." Therefore, the purpose of this study is to look into the origin of the letter designs in t

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.203-214
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• 2000

Porphyromonas gingivalis has been implicated in periodontal diseases. Accumulating evidence suggests that cardiovascular disease is the most prevalent medical problem in patients with periodontal diseases. In order to check the possibility that P. gingivalis is involved in coronary heart disease, the present study was performed to observe P. gingivalis adherence and invasion of human coronary artery endothelial cells (HCAEC) and production of cytokines and growth factors by HCAEC upon P. gingivalis infection. $^3H$-labeled P. gingivalis 381 was incubated with HCAEC for 90 min. The radioactivity of the washed HCAEC was a measure of the absorbed (adhering and invading) P. gingivalis. The absorption radioactivity of the HCAEC infected by P. gingivalis was determined to be 59.58% of the input bacterial cells. In contrast, the absorption radioactivity of the cells infected by S. gordonii Challis which was employed as a control was negligible (0.59%). DPG3, a P. gingivalis mutant defective of fimbriae, appeared to be impaired to some extent in capability of adherence/invasion as compared to that of the parental strain 381, showing 43.04% of the absorption radioactivity. The absorption radioactivity of the HCAEC infected by P. gingivalis 381 in the presence of excessive fimbriae at the concentrations of $50\;{\mu}g$ and $100\;{\mu}g/ml$ was 57.27 and 45.44%, respectively. Invasion of HCAEC by P. gingivalis 381 was observed by an antibiotic (metronidazole) protection assay and transmission electron microscopy (TEM). In the antibiotic protection assay, invasion by the bacterium was measured to be 0.73, 1.09, and 1.51% of the input bacterial cells after incubation for 30, 60, and 90 min, respectively. Invasion by DPG3 was shown to be 0.16% after 90-min incubation. In comparison of invasion efficiency at 90 min of the incubation, the invasion efficiency of DPG3 was 0.37% while that of its parental strain 381 was 2.54%. The immunoblot analysis revealed fimbriae of P. gingivalis did not interact with the surface of HCAEC. These results suggest that fimbriae are not the major contribution to the adherence of P. gingivalis to HCAEC but may be important in the invasion of HCAEC by the bacterium. The presence of cytochalasin D ($1\;{\mu}g/ml$) and staurosporine ($1\;{\mu}M$) reduced the invasion of HCAEC by P. gingivalis 381 by 78.86 and 53.76%, respectively, indicating that cytoskeletal rearrangement and protein kinase of HCAEC are essential for the invasion. Infection of P. gingivalis induced HCAEC to increase the production of TNF-${\alpha}$. by 60.6%. At 90 min of the incubation, the HCAEC infected with P. gingivalis cells was apparently atypical in the shape, showing loss of the nuclear membrane and subcellular organelles. The overall results suggest that P. gingivalis may cause coronary heart disease by adhering to and invading endothelial cells, and subsequently damaging the cells.