• Journal of Embryo Transfer
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• v.14 no.2
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• pp.89-92
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• 1999

효율적인 homozygous 동물을 생산하기 위한 실험의 단계로 염색체가 4배체인 수정란의 이용성을 타진하기 위해 생쥐 수정란과 cytochalasin B를 사용하여 4배체 유도에 관한실험을 수행하였다. 생쥐 2-세포기 수정란을 10$\mu\textrm{g}$/ml 농도의 cytochasin B로 약 20시간 배양하였을 때 모든 수정란은 발육을 거의 멈추었으나, 이 수정란을 cytochalasin B-free medium에 체외배양하였을 때 발육이 재개되어 48시간 후 상실기나 배반포기까지 약 74%의 발육율을 나타내었다. 그러나 발육된 수정란의 세포수는 대조구에 비해 휠신 적은 것으로 나타났다. 염색체 분석결과 cytochalasin B로 처리한 대부분의 수정란은 4배체인 것으로 나타났고 약간의 수정란은 mosaicism과 다배체를 나타내기도 하였다. 그러므로 cytochalasin B를 이용하여 효과적으로 4배체의 수정란을 유도할 수 있는 것으로 나타났다.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.43-49
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• 1997

The objective of this study is to determine developmental pattern and cell allocation to the ICM and TE in haploid and diploid of embryos following parthenogenetic activation and in vitro fertilization. The incidence of development to blastocyst was lower in the combined treatments of ethanol stimulation and cytochalasin B as compared to the control. However, the combined ethanol stimulation and cytochalasin B treatment (diploid) accelerated development to the blastocyst as compared to the ethanol treatment alone (haploid). Significantly reduction in the average number of total cells and ICM was observed in the parthenotes alone as compared to fetilized embryos, but those of combined ethanol stimulation and cytochalasin B treatment embryos were significantly increased as compared to ethanol alone embryos. These results suggested that the ploidy affects preimplantation developmental patters and cell allocation to the ICM and TE in the porcine.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.81-82
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• 2004

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.199-199
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• 2004

This study was conducted to investigate cytoskeleton alterations during vitrified (Open Pulled Straw method) porcine immature oocytes, to utilize Taxol/sup TM/ (polymerization of tubulin molecules) and Cytochalasin B (CB, depolymerization of actin filaments) during vitrification to stabilize microtubule and microfilaments (MT and MF), and to determine in vitro maturation, fertilization and development of cytoskeletal-stabilized and vitrified porcine immature oocytes. (omitted)

• Development and Reproduction
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• v.9 no.1
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• pp.49-52
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• 2005

The effects of cytochalasin B was studied for the cleavage and development of in vitro matured porcine follicular oocytes. The follicular oocytes were collected from slaughtered pig ovaries and matured for 65 hours. The matured oocytes were activated by 7% ethanol(v/v) in DPBS and the activated oocytes were subjected to cytochalasin B concentrations of 2.5, 5.0 and $7.5\;{\mu}g/mL$ for 3, 5 and 7 hours, and then the treated oocytes were cultured in NCSU23 with 0.4% BSA for 7 days. The cleavage rates were not different significantly in each treatment. However, the oocytes treated with $5.0\;{\mu}g/mL$ for 5 hours yielded a significantly higher morula rate(19.7%) than oocytes treated with $2.5\;{\mu}g/mL$ for 3 and 5 hours(9.4%). The sum rate of $2.5\;{\mu}g/mL$ concentration(10.5%) by hour was also significantly lower than those of 5.0(18.0%) and $7.5\;{\mu}g/mL$ concentration(14.6%). The blastocyst rate in oocytes treated with $5.0\;{\mu}g/mL$ for 3(9.4%) and 5 hours(9.0%) was significantly higher than the rate in oocytes treated with $2.5\;{\mu}g/mL$ for 3 hours(0%). The sum rate of $5.0\;{\mu}g/mL$ concentration also significantly higher than those of 2.5 and $7.5\;{\mu}g/mL$ concentration. The results demonstrated that the treatment of oocytes with cytochalasin B of $5.0\;{\mu}g/mL$ for $3{\sim}5$ hours was the optimal concentration and duration for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes.

• Biomedical Science Letters
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• v.9 no.4
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• IMMUNE NETWORK
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• v.11 no.6
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• pp.424-427
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• 2011

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-$1{\beta}$ and TNF-${\alpha}$ was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-${\kappa}B$ activation and cytokine expression. Treatment with M. leprae significantly increased NF-${\kappa}B$ activation and expression of TNF-${\alpha}$ and IL-$1{\beta}$ in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.229-236
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• 2002

Objective : The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. Methods : Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at $37^{circ}C$ for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at $4^{circ}C$ overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. Results: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05). Conclusion: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

• Journal of Life Science
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• v.24 no.1
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• pp.86-91
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• 2014

Trichoplusia ni cells are used as a host permissive cell line in the baculovirus expression system, which is useful for large-scale production of human sugar transport proteins. However, the activity of endogenous sugar transport systems in insect cells is extremely high. Therefore, the transport activity resulting from the expression of exogenous transporters is difficult to detect. Furthermore, very little is known about the nature of endogenous insect transporters. To exploit the expression system further, the effect of D-fructose on 2-deoxy-D-glucose (2dGlc) transport by T. ni cells was investigated, and T. ni cell-expressed human transporters were photolabeled with [$^3H$] cytochalasin B to develop a convenient method for measuring the biological activity of insect cell-expressed transporters. The uptake of 1 mM 2dGlc by uninfected- and recombinant AcMPV-GTL infected cells was examined in the presence and absence of 300 mM of D-fructose, with and without $20{\mu}M$ of cytochalasin B. The sugar uptake in the uninfected cells was strongly inhibited by fructose but only poorly inhibited by cytochalasin B. Interestingly, the AcMPV-GTL-infected cells showed an essentially identical pattern of transport inhibition, and the rate of 2dGlc uptake was somewhat less than that seen in the non-infected cells. In addition, a sharply labeled peak was produced only in the AcMPV-GTL-infected membranes labeled with [$^3H$] cytochalasin B in the presence of L-glucose. No peak of labeling was seen in the membranes prepared from the uninfected cells. Furthermore, photolabeling of the expressed protein was completely inhibited by the presence of D-glucose, demonstrating the stereoselectivity of labeling.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.109-113
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• 2007

This study was conducted to evaluate the effect of cytochalasin B (CB) treatment in the activation medium on the development of somatic cell nuclear transfer (SCNT) rat embryos. Fetal fibroblast cells were isolated from a Day 14.5 fetus, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ with or without CB for 4 hr, and formation of pseudo-pronucleus (PPN) was checked at 18 hr after activation. Then, they were transferred into day 1 pseudopregnant recipients (Hooded Wistar) or cultured for 5 days to check their developmental competence in vivo or in vitro. The number of PPN was not affected by CB treatment during the activation. However, CB treatment supported pre-implantation development of rat SCNT embryos. Embryos generated by the procedures of SCNT were also capable of implanting, with 1 implantation scar found from a recipient following the transfer of 87 SCNT embryos to four foster mothers. The result of the present study shows that rat SCNT embryo can develop to post-implantation stage following treatment with CB.