• ALGAE
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• 제25권4호
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• pp.197-204
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• 2010

Cytoskeletal changes were observed during cell division of the green alga Zygnema cruciatum using flourescein isothiocynate (FITC)-conjugated phallacidin for F-actin staining and FITC-anti-$\alpha$-tubulin for microtubule staining. Z. cruciatum was uninucleate with two star-shaped chloroplasts. Nuclear division and cell plate formation occurred prior to chloroplast division. Actin filaments appeared on the chromosome and nuclear surface during prophase, and the F-actin ring appeared as the cleavage furrow developed. FITC-phallacidin revealed that actin filaments were attached to the chromosomes during metaphase. The F-actin ring disappeared at late metaphase. At telophase, FITC-phallacidin staining of actin filaments disappeared. FITC-anti-$\alpha$-tubulin staining revealed that microtubules were arranged beneath the protoplasm during interphase and then localized on the nuclear region at prophase, and that the mitotic spindle was formed during metaphase. The microtubules appeared between dividing chloroplasts. The results indicate that a coordination of actin filaments and microtubules might be necessary for nuclear division and chromosome movement in Z. cruciatum.

• Applied Microscopy
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• 제33권2호
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• pp.105-116
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• 2003

U. unicinctus 난세포를 인공수정한 결과 수정 시기는 germinal vesicle (2n)기였으며 정자 중앙부에서 돌출한 actomere와 난모세포의 미세융모 끝에서 세포막 융합이 시작되는 것으로 관찰되었다. Germinal vesicle기에 수정이 가능하였으므로 pre-mitotic spindle이 관찰되지 않은 것으로 사료되었다. 수정 후 난모세포는 제1, 2감수분열을 수행하였으며 각각의 감수분열 장치들은 감수분열 말기에 난모세포막과 밀접한 구조를 형성하였고, 이 부위에서 극체가 형성되는 것을 볼 수 있었다. 극체 형성시 난모세포막은 세포분열의 관점에서 형태가 없는 것이 아니라 항-튜블린-FITC에 대한 활성구조를 형성하였으며, 각 감수분열 장치의 한쪽 극 (pole)과 어떤 복합구조를 형성하는 것으로 보인다. 제2극체 형성도 제1극체와 유사한 방법으로 형성되었으나, 제2감수분열 장치는 난모세포막의 접선에 평행하게 생성된 후 세포막을 향해 이동하면서 방추사의 양극성이 회전 (shift-rotation)하였고 접선에 수직으로 정렬하는 것을 볼 수 있었다. 난모세포의 감수분열 장치에서 방추사의 활성은 강하였으나 aster의 활성은 비교적 약한 것으로 보였다. 제2감수분열이 진행되는 동안 난모세포질에 머믈고 있던 정자는 점차 미래의 자성 전핵 형성부인 난모세포막 근접부로 이동하는 것이 관찰되었다. 난모세포막 근접부로 이동하는 동안 정자 aster의 활성은 점차 강해지는 반면 난모세포의 aster에서는 활성이 미약한 것으로 보아서 자웅 전핵융합을 주도하는 것은 정자 유래의 aster인 것으로 사료되었다. 제1난모세포의 감수분열 장치가 활성화되는 시기에 제1극체의 방추사에서도 강한 활성을 관찰할 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Journal of Microbiology and Biotechnology
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• 제30권2호
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• pp.279-286
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• 2020

A novel compound named 'kanakugiol' was recently isolated from Lindera erythrocarpa and showed free radical-scavenging and antifungal activities. However, the details of the anti-cancer effect of kanakugiol on breast cancer cells remain unclear. We investigated the effect of kanakugiol on the growth of MCF-7 human breast cancer cells. Kanakugiol affected cell cycle progression, and decreased cell viability in MCF-7 cells in a dose-dependent manner. It also enhanced PARP cleavage (50 kDa), whereas DNA laddering was not induced. FACS analysis with annexin V-FITC/PI staining showed necrosis induction in kanakugiol-treated cells. Caspase-9 cleavage was also induced. Expression of death receptors was not altered. However, Bcl-2 expression was suppressed, and mitochondrial membrane potential collapsed, indicating limited apoptosis induction by kanakugiol. Immunofluorescence analysis using α-tubulin staining revealed mitotic exit without cytokinesis (4N cells with two nuclei) due to kanakugiol treatment, suggesting that mitotic catastrophe may have been induced via microtubule destabilization. Furthermore, cell cycle analysis results also indicated mitotic catastrophe after cell cycle arrest in MCF-7 cells due to kanakugiol treatment. These findings suggest that kanakugiol inhibits cell proliferation and promotes cell death by inducing mitotic catastrophe after cell cycle arrest. Thus, kanakugiol shows potential for use as a drug in the treatment of human breast cancer.