• 생명과학회지
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• 제28권8호
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• pp.885-891
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• 2018

급성위막성대장염(Pseudomembranous colitis)은 C. difficile 세균이 분비하는 톡신A에 의해 유발되는 것으로 알려져 있다. 톡신A에 의한 점막 상피세포의 장벽기능 감소가 발병 원인으로 알려져 있다. 최근 연구에 의하면 톡신 A는 대장상피세포 속 HDAC-6의 활성을 높여 튜블린의 탈아세틸화를 증가시키는 것으로 알려져 있다. 튜블린 단백질의 탈아세틸화는 미세소관 불 형성을 초래하여 점막 상피세포의 극단적인 세포 형태 변형을 야기하게 되며 결국 상피세포의 고유기능인 장벽 기능이 파괴된다고 알려져 있다. 최근 연구자 등은 potassium acetate가 톡신A에 의한 튜블린 탈아세틸화와 미세소관 불 형성을 회복시켜 장염을 유의하게 억제함을 보고하였다. 따라서 본 연구에서는 아세틸기를 포함하는 또 다른 간단한 화학구조의 초산을 적용하여 톡신A의 세포독성을 억제하는지 확인해보고자 하였다. 인간 대장상피세포에서 초산 자극은 튜블린 단백질의 아세틸화를 유의하게 증가시켰다. 또한 초산은 대장상피세포 속 미세소관 형성과정도 강하게 촉진시킴을 확인하였다. 초산은 톡신A에 의한 튜블린 탈아세틸화와 미세소관 불 형성 그리고 세포독성 모두를 유의하게 회복시켰다. 이상의 결과는 초산에 의한 미세소관 형성 촉진이 톡신A에 의해 초래되는 세포골격계 파괴와 그로 인한 세포독성을 억제할 수 있음을 보여준다. 따라서 초산이 톡신A의 작용을 차단하여 위막성대장염 증상을 완화시킬 수 있는 치료제로서 개발 가치가 있음을 보여준다.

• Archives of Pharmacal Research
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• 제25권2호
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• pp.191-196
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• 2002

The water extract from the root bark of Cortex Mori (CM, Morus alba L.: Sangbaikpi), a mulberry tree, has been known in Chinese traditional medicine to have antiphlogistic, diuretic, and expectorant properties. In this study, the cytotoxicity of CM against tumor cells and its mechanism was examined . CM exhibited cytotoxic activity on K-562, B38O human leukemia cells and B16 mouse melanoma cells at concentrations of > 1 mg/ml. A DNA fragmentation, PARP cleavage, and nuclear condensation assay showed that those cells exposed to CM underwent apoptosis. The water extract of Scutellarie Radix (SR) was used as a negative control and showed no cytotoxicity in those cells. The flow cytometric profiles of the CM-treated cells were also indicative of apoptosis. However, they did not appear to exert the G1 arrest, which is observed in other tubulin inhibitor agents such as vincristine, taxol. The protein-binding test using Biacore and a microtubule assembly-disassembly assay provided evidence showing that CM bound to the tubulins resulting in 3 markets inhibition of the assembly, but not the disassembly of microtubules. The possible nonspecific effect of the CM extract could be excluded due to the results using SR, which did not affect the assembly process. Overall, the water extract of CM induces apoptosis of tumor cells by inhibiting microtubule assembly.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.13-18
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• 2009

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing $\beta$-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

• Molecules and Cells
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• 제27권1호
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• pp.1-3
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• 2009

Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle act as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.

• 대한의생명과학회지
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• 제15권1호
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• pp.67-72
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• 2009

Microtubule-interfering agents (MIAs), including paclitaxel, have been attributed in part to interference with microtubule assembly, impairment of mitosis, and changes in cytoskeleton. But the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. This study investigated the effect of paclitaxel on differentiation such as type II collagen expression and sulfated proteoglycan accumulation in rabbit articular chondrocytes. Paclitaxel caused differentiated chondrocyte phenotype as demonstrated by increment of type II collagen expression and proteoglycan synthesis Paclitaxel treatment stimulated activation of ERK-1/2 and p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced paclitaxel-induced differentiation, whereas inhibition of p38 kinase with SB203580 suppressed paclitaxel-induced differentiation. Our findings suggest that ERK-1/2 and p38 kinase oppositely regulate paclitaxel-induced differentiation in chondrocytes.

• 미생물학회지
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• 제45권2호
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• pp.228-231
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• 2009

고추 역병을 야기하는 Phytophthora capsici 는 짧은 시간 내에 많은 면적에 피해를 주는 병으로 한번 발생하면 방제가 어려운 병으로 알려져 있다. 이러한 역병 곰팡이의 방제를 위하여 본 연구에서는 P. capsici의 염색체 복제 및 세포 골격 유지 등에 관여하는 단백질인 microtubule의 형성 저해를 유도하는 물질을 탐색하여 궁극적으로 고추역병 방제를 위한 연구를 진행하였다. 먼저 P. capsici alpha 및 beta tubulin을 E. coli BL21(DE3)에서 발현시켜 분리 정제하여 in vitro microtubule 형성을 확인하였다. P. capsici microtubule 형성 저해 metagenome clone 스크리닝을 위하여 경기도 수원의 여기산 토양에서 metagenome을 분리하여 library를 제작하여 Fluorescence Resonance Energy Transfer (FRET) 방법을 이용하여 P. capsici microtubule 형성을 저해하는 화합물을 탐색하였다. In vitro 스크리닝에서 약 384개의 metagenome library에서 2종의 clone을 선택하여 고추작물에 직접 방제하여 역병균의 생장 억제를 확인하였다. 이는 차후 고추역병 방제제 개발에 있어 중요한 후보물질뿐만 아니라 metagenome library를 이용한 새로운 방법의 개발이라 사료 된다. 또한 in vitro 스크리닝에서 얻어진 2종의 metagenome clone의 염기서열을 분석하여 항역병 활성에 관련하는 DNA 서열을 확보하고 이를 응용하여 물질을 생산 할 경우, 현장에서 활용 할 수 있는 효과 큰 친환경 천연고추역병 방제제로서의 개발 가능성을 가진다는 점에서 본 연구결과는 매우 의미 있는 결과라 생각된다.

• BMB Reports
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• 제40권2호
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• pp.218-225
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• 2007

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation exp eriments demonstrated that AtMAP65-1 bound to tubulin dimers,at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with $\alpha$-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 $\Delta$N339 (amino acids 340-587); AtMAP65-1 $\Delta$N494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 $\Delta$C495 (amino acids 1-494) and AtMAP65-1 $\Delta$C340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 $\Delta$N339, $\Delta$N494, AtMAP65-1 340-494 and $\Delta$C495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 $\Delta$N339, $\Delta$N494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 $\Delta$N339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 $\Delta$N494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 $\Delta$C495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.51-51
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• 2002

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we compared chromatin, r-tubulin and microtubule organization in porcine oocytes following somatic cell nuclear transfer and parthenogenetically activation in order to clarify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. (omitted)

• Reproductive and Developmental Biology
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• 제34권2호
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• pp.73-79
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• 2010

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing $\gamma$-tubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The $\gamma$-tubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas $\gamma$-tubulin spots in the SCNT embryos were observed only during the inter-anaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.693-699
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• 2016

Clostridium difficile toxin A is known to cause deacetylation of tubulin proteins, which blocks microtubule formation and triggers barrier dysfunction in the gut. Based on our previous finding that the Clostridium difficile toxin A-dependent activation of histone deacetylase 6 (HDAC-6) is responsible for tubulin deacetylation and subsequent microtubule disassembly, we herein examined the possible effect of potassium acetate (PA; whose acetyl group prevents the binding of tubulin to HDAC-6) as a competitive/false substrate. Our results revealed that PA inhibited toxin A-induced deacetylation of tubulin and recovered toxin A-induced microtubule disassembly. In addition, PA treatment significantly decreased the production of IL-6 (a marker of inflamed tissue) in the toxin A-induced mouse enteritis model. An in vitro HDAC assay revealed that PA directly inhibited HDAC-6-mediated tubulin deacetylation, indicating that PA acted as a false substrate for HDAC-6. These results collectively indicate that PA treatment inhibits HDAC-6, thereby reducing the cytotoxicity and inflammatory responses caused by C. difficile toxin A.