• Journal of Life Science
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• 제10권2호
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• pp.39-44
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• 2000

Trichostatin A (TSA) is a Streptomyces product, which inhibits the enzyme activity of histone deacetylase. It is also known as an inducer of apoptosis in several human cancer cell lines. In this study, we investigated the mechanism of apoptosis induced by TSA in MDA-MB-231 human breast carcinoma cells. The cytotoxicity of TSA on MDA-MB-231 cells was assessed by MTT assay. The cell viability was decreased dose-dependently and the IC\ulcorner value was about 100 ng/ml after 48 h treatment with TSA. Morphological change and DNA ladder formation, the biochemical hallmarks of apoptotic cell death, were observed after treatment of TSA in a concentration-dependent manner, which was accompanied with cleavage of poly(ADP-ribose) polymerase and $\beta$-catenin, and activation of caspase-3. TSA treatment up-regulated the expression of a cyclin-dependent kinase inhibitor p21 (Wafl/Cip1) protein, a key regulatory protein of the cell cycle. However, there is no detectable change of both Bcl-2 and Bax expressions. These results demonstrated that TSA might inhibit cell growth through apoptosis in human breast carcinoma MDA-MB-231 cells.

• Molecular & Cellular Toxicology
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• 제5권3호
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• pp.222-229
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• 2009

Our previous finding that pre-initiation treatment of indole-3-carbinol (I3C) represents a chemopreventive effect in dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis has prompted us to test the global expression of genes at an early stage. Rats were continuously fed 300 ppm I3C in their diet at 6 weeks of age and were injected with DMBA at 7 weeks of age, and were sacrificed at 8 weeks of age. Global gene expression analysis using oligonucleotide microarrays was conducted to detect altered genes in DMBA- or DMBA plus I3C-treated mammary glands. Altered genes were identified by fold changes of 1.2 and by t-test (P<0.05) from the log ratios of the hybridization intensity of samples between control (Group 1) and DMBA (Group 2), and from those of samples between DMBA (Group 2) and DMBA plus I3C (Group 3). From these genes, we chose altered genes that were up- or down-regulated by DMBA treatment and recovered to the control level by I3C treatment. For early stage of carcinogenesis, I3C treatment induced the recovery to normal levels of several genes including cell cycle pathway (cyclin B2, cell division cycle 2 homolog A), MAP signaling pathway (fibroblast growth factor receptor 1, platelet derived growth factor receptor, beta polypeptide), and insulin signaling (protein phosphatase 1, regulatory (inhibitor) subunit 3B and flotillin 2), which were up-regulated by DMBA treatment. In addition, I3C treatment induced the recovery to normal levels of several genes including those of MAPK signaling (transforming growth factor, beta receptor 1 and protein phosphatase 3, catalytic subunit, beta isoform), which were down-regulated by DMBA treatment. These results suggest that the targeting of these genes presents a possible approach for chemoprevention in DMBA-induced mammary carcinogenesis.

• Journal of Microbiology
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• 제35권1호
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• pp.61-65
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• 1997

A novel strategy was developed for isolating suppressors from sporulation-deficient mutants. The mutation in the BCY1 gene, which codes for the regulatory subunit of cAMP-dependent protein kinase, when homozygous, results in diploids being meiosis and sporulation deficient. Two plasmids, YCp-MAT.alpha. and YEp-SPOT7-lacZ, were introduced into MAT.alpha. BCY1$\^$+/ or MAT.alpha. bcy1 haploid cells. The transformant of the BCY1$\^$+/ haploid cell produced .betha.-galactosidase under nutrient starvation, but the bcy1 transformant did not. Using this system, the mutagenesis experiment performed on the bcy1 transformant strain resulted in a number of sporulation mutants that produced .betha.-galactosidase under nutrient starvation. One complementation group, sob1, was identified from the isoalted suppressor mutants and characterized as a single recessive mutation by tetrad analysis. Genetic analysis revealed that the sob1 mutation suppressed the sporulation deficiency, the failure to arrest at the G1 phase of the cell cecle, and the sensitivity to heat or nitrogen starvation caused by the bcy1 mutation. However, the sob1 mutation did not suppress the sporulation deficiency of ime1 and of ime2 diploids. These results suggest that the sob1 mutation affects a gene which functions as a downstream regulator in both meiosis and cell cycle regulation.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.323.3-324
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• 2002

Histone deacetylases (HDAC) activity is associated generally with transcriptional repression. We have reported previously that apicidin. a histione deacetylase inhibitor. inhibited the proliferation of tumor cells via induction of p21 WAF/C1P1. We extended our study to identify the effect of apicidin on the expression of other cell cycle regulatory protein. such as cyclin E. a critical regulator of the transition from G1 into S phase. (omitted)

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.27-27
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• 2003

A diversified and concentrative approach of methylation player can be one of the most powerful studies in the understanding of global epigenetic modifications. Previous studies have suggested that DNA methylation contributes to transcriptional silencing through the several DNA methylation-mediated repression systems by hypermethylation, including methyltransferases (DNMTs), DNA methyltransferase association protein 1 (DMAPl), methyl-CpG binding domain (MBD), and histone deacetylases (HDACs). Assembly of these regulatory protein complexes act sequentially, reciprocally, and interdependently on the newly composed DNA strand through S phase. Therefore, these protein complexes have a role in coupling DNA replication to the designed turn-off system in genome. In this study, we attempted to address the role of DNA methylation by the functional analysis of the methyltransferase molecule, we described the involvement of DMAP1 and DNMTs in cell divistion and the effect of their loss. We also described distinct patterns that DMAP1 and DNMTs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis in HeLa cell, COS7, and HIH3T3 cell cycle progressions. DNMT1, DNMT3b, and DMAP1 do not stably contact the genetic material during chromosome compaction and repressive expression. These finding show that the loss of activities of DNMTs and DMAP1 occure stage specifically during the cell cycle, may contribute to the integral balance of global DNA methylation. This is consistent with previous studies resulted in decreased histone acetyltransferases and HDACs, and differs from studies resulted in increased histone methyltransferases. Our results suggest that DNA methylation by DNMTs and DMAP1 during mitosis acts to antagonize hypermethylation by which this mark is epigenetical mitotic-specific methylation.

• 대한골관절종양학회지
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• 제14권2호
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• pp.106-118
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• 2008

목적: 세포주기의 조절곤란은 종양의 발생과 진행에 영향을 미친다. 몇가지 알려진 연부조직 육종에 대한 면역 조직화학적 예후인자 들이 있지만 상반된 연구도 있고 G1/S phase관계되는 인자에 대한 연구는 거의 없는 상태이다. 따라서 저자는 연부조직 육종의 재발 및 전이와 관계된 G1/S phase 세포조절주기 단백의 면역화학적 예후인자를 알아보고자 하였다. 대상 및 방법: 1998년 1월부터 2005년 12월까지 연부조직 육종으로 진단된 환자 중 최소한 1년이상 추시관찰이 가능하고 파라핀 블록의 보존상태가 비교적 양호한 43예의 환자를 대상으로 연구하였다. 지방육종 15예, 악성 섬유성조직구증 13예, 횡문근육종 5예, 활막육종 5예, 평활근육종 3예, 섬유육종이 2예였다. 모든 환자의 조직은 전 절제술로 제거된 조직을 대상으로 Cyclin D1, Cyclin E, CDK4, CDK, p16, p27, Rb, E2F-1, p53, Ki-67 등의 면역 조직화학적 발현을 조직 microarray 방법을 사용하여 측정하고 환자의 국소 재발 및 전이에 따른 예후를 비교 분석하였다. 결과: 국소 재발은 8예(19%)에서 일어났으며 이것은 Cyclin E(p=0.024), E2F-1(p=0.046)의 발현과 연관이 있었다. 전이는 16예(37%)에서 일어났으며 CDK4의 증가와 연관이 있었다(p=0.031). 결론: Cyclin E와 E2F-1이 연부조직 육종의 국소 재발과 관련있는 예후를 제공해주었고, CDK4가 전이를 예측할 수 있는 독립적인 예후를 제공해주었다. 따라서 조직 검사시 이들 표식자들의 적절한 사용이 환자의 예후를 예측하고 치료의 범위를 결정 짖는데 중요한 도움을 줄 수 있을 것이다.

• 미생물과산업
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• 제25권1호
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• pp.2-9
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• 1999

A study was made on enzymes of carbohydrate metabolism in T. concretivorus grown with and without glucose. The present results show that T. concretivorus possesses high activities of pentose shunt pathway and related enzymes, glucokinase, G-6-P dehydrogenase, 6-PG dehydrogenase, and phosphoglucoisomerase, but low activities of enzymes unique to EMP(fructose-1,6-diphosphate aldolase). Although the synthesis of the latter enzymes remains largely unaffected by the growth enviroment, that of the former is stimulated by glucose. And the failure to detect ED pathway enzymes in cells grown in thiosulate or thiosulfate-glucose medium eliminates the ED pathway as a significant route of glucose catabolism in T.concretivorus. These results suggest that pentose shunt pathway performs an energetic role in glucose metabolism by T.concretivorus with EMP as a subway. The absence of ED pathway and the presence of pentose shunt pathway which is the major route of catabolism in T.concretivorus are similar to those of other obligately chemolitho-trophic thiobacilli. The G-6-P and 6-PG dehydrogenase are both NAD and NADP specific, but MAD predominant. However, the 3-PGAL dehydrogenase is only NAD specific. Since the specific activity of 3-PGAL generated from glucose is converted mainly into pyruvate which is channeled into the TCA cycle. All enzymes of the TCA cycle tested and NADH oxidase are detected in the cells of T.concretivorus grown in thiosulfate. The specific activities of fumarase and isocitrate dehydrogenase are high and others are low. The presence of two isocitrate dehydrogenase (NAD-and NADP-linked) may have important regulatory function for this organism. The activity of NAD-oxidase, which is implicated in the energy generating metabolism, was very high in the crude cell-free extract of T.concretivorus, recording 55.11 m.mu. mole/min/mg protein. This well coincides with the fact that activities of NAD-linked G-6-P dehydrogenase, 6-PG dehydrogenase and 3-PGAL dehydrogenase were high.

• International Journal of Industrial Entomology and Biomaterials
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• 제22권2호
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• pp.59-64
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• 2011

Protein kinase C (PKC) is involved in many cellular signaling pathways, it participates in many physiological processes, such as cell cycle, growth, proliferation, differentiation and apoptosis. To investigate the effect of PKC on the silkworm midgut tissue infection of Bombyx mori parvo-like virus (BmPLV), a B. mori atypical protein kinase C (BmaPKC) gene was cloned from larval midgut tissue, expressed in E. coli and purified. Additionally, the BmPLV susceptible silkworm strain and resistant silkworm strain were used to test the effect of the B. mori infection on BmPLV. The result showed that BmaPKC encodes a predicted 586 amino acid protein, which contains a C-terminal kinase domain and an N-terminal regulatory domain. The maximum expression amount of the soluble (His)6-tagged fusion protein was detected after 0.8 mmol/L IPTG was added and cultured at $21^{\circ}C$. The (His) 6-tagged fusion protein revealed about 73 kDa molecular weight which confirmed by western blot and mass spectrography. Furthermore BmaPKC protein were detected at 0-72 h post-infection in BmPLVinfected larval midgut tissue, western blot showed that as time went on, the expression of BmaPKC increased gradually in susceptible strain, the expression quantity on 72 h is 5 times of 0 h. However, in resistant strain, the expression quantity is slightly lower than susceptible strain. But no significant change in resistant strain was observed as time went on. The available data suggest that BmaPKC may involve in the regulation of BmPLV proliferation.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.235-240
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• 2002

방선균은 토양속에 서식하는 그람 양성 세균으로 세포성 장의 어느 시기에 영양세포가 이어져 연쇄상의 기균사를 형성하고 그 끝에 포자를 형성하는 동시에 생리학적 분화로 표현되는 다양한 이차대사물질을 생산한다. 이들의 복잡한 생활사에 따른 분화에는 진핵생물의 ser/thr protein kinase와 원핵생물의 his/asp acid protein kinase 등과 같은 다양한 신호전달 단백질들이 조절을 담당하고 있다. Akt kinase는 진핵생물에서 보고된 ser/thr kinase로.세포내의 다양한 신호전달기구를 조절하고 있으며, 세포내의 Akt kinase의 활성화 또는 불활성화가 세포 증식, 분화, 생존, 세포사등의 신호전달에 결정적인 역할을 담당한다. 방선균으로부터 Akt kinase와 유사한 기능을 갖는 신호전달 단백질을 규명하기 위하여, Akt kinae의 target단백질들의 인산화 부위 보존영역으로부터 나타나는 아미노산의 consensus sequence를 기초로 하여 형광물질로 라벨시킨 합성 peptide(FITC-TRRSRfESIT)를 제작하였다 제작한 기질 peptide에 인산화가 일어나면 아가 로스 전기영동상에서의 운동성에 차이가 나타나고, 이를 자외선하에서 형광 peptide를 관찰하는 방법으로 인산화 assay를 실시하였다. S. griseus IFO 13350을 배양한 cell-free extract로부터 ammonium sulfate fractionation과 DEAE-Sepharose, Mono Q, Resource Phenyl-Superose, Gel permeation 등 수 단계의 column chromatography를 통하여 Akt 유사 단백질을 정제하였다. 그 결과 방선균에도 고등생 물의 Akt와 유사한 기질특이성을 갖는 인산화 단백질이 존재하는 것으로 판단되었으며, 그 중의 하나는 분자량이 39 kDa 정도의 크기를 갖는 단백질로 판명되었다. 지금까지의 인산화 단백질 연구는 활성측정법이 어려워 연구자들에게 많은 제한을 주어 왔지만, 본 연구에서 사용한 합성 peptide를 이용하는 방법을 보다 다양한 인산화 단백질에 대하여 적용한다면, 인산화 단백질 및 조절물질 개발에 많은 도움이 될 수 있을 것으로 예상된다.

• BMB Reports
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• 제44권3호
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• pp.147-157
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• 2011

The meiotic process from the primordial stage to zygote in female germ cells is mainly adjusted by post-transcriptional regulation of pre-existing maternal mRNA and post-translational modification of proteins. Several key proteins such as the cell cycle regulator, Cdk1/cyclin B, are post-translationally modified for precise control of meiotic progression. The second messenger (cAMP), kinases (PKA, Akt, MAPK, Aurora A, CaMK II, etc), phosphatases (Cdc25, Cdc14), and other proteins (G-protein coupled receptor, phosphodiesterase) are directly or indirectly involved in this process. Many proteins, such as CPEB, maskin, eIF4E, eIF4G, 4E-BP, and 4E-T, post-transcriptionally regulate mRNA via binding to the cap structure at the 5' end of mRNA or its 3' untranslated region (UTR) to generate a closed-loop structure. The 3' UTR of the transcript is also implicated in post-transcriptional regulation through an association with proteins such as CPEB, CPSF, GLD-2, PARN, and Dazl to modulate poly(A) tail length. RNA interfering is a new regulatory mechanism of the amount of mRNA in the mouse oocyte. This review summarizes information about post-transcriptional and post-translational regulation during mouse oocyte meiotic maturation.