• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.719-723
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• 2014

Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.139-145
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• 2009

The purpose of this study was to evaluate the effects of the water extract of Samultang (SMT), a Chinese herb, on apoptotic cell death by $H_2O_2$-induced oxidative stress in SK-N-M C cells. A nuclear fragmentation was observed via fluorescence imaging 12 h after exposure to 30 ${\mu}M$ $H_2O_2$ and DNA laddering was detected via agarose electrophoresis gel. In addition, increases in sub-G1 phase and cleavage of the PARP protein were observed. However, treatment with SMT for 2 h prior to $H_2O_2$ exposure significantly reduced apoptotic cell death induced by incubation with 30 ${\mu}M$ $H_2O_2$ in SK-N-MC cells. Pre-incubation with water extract of SMT for 2 h prevented the $H_2O_2$-induced decrease in mitochondrial transmembrane potential. SMT also attenuated the increase in caspase-3 activity and the breakdown of PARP protein caused by $H_2O_2$-induced oxidative stress. These results suggest that the water extract of SMT provides inhibition of apoptotic cell death against oxidative injury in SK-N-MC cells.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7537-7542
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• 2013

Background: Terpinen-4-ol, a monoterpene, is found as the main component of essential oil extracts from many plants. In this study apoptotic and autophagic types of cell death induced by terpinen-4-ol and associated mechanisms were investigated in human leukemic HL-60 cells. Materials and Methods: The cytotoxicity of human leukemic U937 and HL-60 cells was determined by MTT assay. Cytochrome c release, expression of Bax, Bcl-2, Bcl-xl and cleaved Bid were determined by Western blotting. Cell morphology was examined under a transmission electron microscope. LC3-I/II, ATG5 and Beclin-1 levels were detected by immunoblotting. Results: Terpinen-4-ol exhibited cytotoxicity to human leukemic HL-60 but not U937 cells. The apoptotic response to terpinen-4-ol in HL-60 cells was due to induction of cytochrome c release from mitochondria and cleavage of Bid protein after the stimulation of caspase-8. There was a slightly decrease of Bcl-xl protein level. The characteristic cell morphology of autophagic cell death was demonstrated with multiple autophagosomes in the cytoplasm. At the molecular level, the results from Western blot analysis showed that terpinen-4-ol significantly induced accumulation of LC3-I/II, ATG5 and Beclin-1, regulatory proteins required for autophagy in mammalian cells. Conclusions: Terpinen-4-ol induced-human leukemic HL-60 cell death was via both autophagy and apoptosis.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.599-602
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• 2005

Truncated form of UBP1, an ubiquitin-specific protease of Saccharomyces cerevisiae, was overexpressed in Escherichia coli. The hexahistidine residue $(His_6)$ was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were $40^{\circ}C$ and pH 8.0, respectively.

• Molecules and Cells
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• 제41권11호
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• pp.964-970
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• 2018

Atherosclerosis preferentially involves in prone area of low and disturbed blood flow while steady and high levels of laminar blood flow are relatively protected from atherosclerosis. Disturbed flow induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). ER stress is caused under stress that disturbs the processing and folding of proteins resulting in the accumulation of misfolded proteins in the ER and activation of the UPR. Prolonged or severe UPR leads to activate apoptotic signaling. Recent studies have indicated that disturbed flow significantly up-regulated $p-ATF6{\alpha}$, $p-IRE1{\alpha}$, and its target spliced XBP-1. However, the role of laminar flow in ER stress-mediated endothelial apoptosis has not been reported yet. The present study thus investigated the role of laminar flow in ER stress-dependent endothelial cell death. The results demonstrated that laminar flow protects ER stress-induced cleavage forms of PARP-1 and caspase-3. Also, laminar flow inhibits ER stress-induced $p-eIF2{\alpha}$, ATF4, CHOP, spliced XBP-1, ATF6 and JNK pathway; these effects are abrogated by pharmacological inhibition of PI3K with wortmannin. Finally, nitric oxide affects thapsigargin-induced cell death in response to laminar flow but not UPR. Taken together, these findings indicate that laminar flow inhibits UPR and ER stress-induced endothelial cell death via PI3K/Akt pathway.

• Preventive Nutrition and Food Science
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• 제7권2호
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• pp.128-132
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• 2002

The present study was performed to observe the role of protein kinase C (PKC) inhibitors (H-7, staurosporine) and daunorubicin in the cell death process of MCF7 cells; and examined whether or not the type of induced cell death was apoptosis. The usefulness of the combined therapy of PKC inhibitors and daunorubicin to improve the adverse effect of daunorubicin was also investigated. Cell death was induced by treatment with PKC inhibitors or daunorubicin. Characteristic morphologic features of cell shrinkage, chromatic condensation, and cytoplasmic vacuolization were observed. These treatments also stimulated the cleavage of poly-(ADP-ribose) polymerase (PARP), an early event in apoptosis. With slight differences in the percentage of apoptosis-induced cells, staurosporine, H-7 and daunorubicin effectively induced apoptosis in MCF7 cells. Furthermore, combined treatment of PKC inhibitors and daunorubicin significantly drove the cells into an apoptotic state. Hence, our results revealed the possible therapeutic value of combined therapy for the prevention of drug resistance and adverse side effects.

• BMB Reports
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• 제44권6호
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• pp.381-386
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• 2011

In the present study, we characterized the function of HS1-binding protein 3 (HS1BP3), which is mutated in essential tremor and may be involved in lymphocyte activation. We found that HS1BP3 localized to the mitochondria and endoplasmic reticulum partially. Overexpression of HS1BP3 induced apoptosis in HEK293T and HeLa cell lines. When these cell lines were transfected with HS1BP3, they exhibited nuclear DNA condensation, externalization of phosphatidylserine (PS), and cleavage of poly ADP ribose polymerase (PARP). Furthermore, suppression of HS1BP3 or HS1 expression attenuates HS1BP3 induced apoptosis. In addition, HS1BP3 enhanced activator protein 1 (AP-1)-mediated transcription in a dose-dependent manner. Therefore, we conclude that HS1BP3 regulates apoptosis via HS1 and stimulates AP-1-mediated transcription.

• International Journal of Industrial Entomology and Biomaterials
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• 제27권2호
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• pp.228-236
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• 2013

Breast cancer is one of the most common cancers among women worldwide. Recently anticancer agents have been developed using natural substances. To evaluate the anticancer effect of hydrolysates of silk fibroin (HSF), we investigated the effect of HSF on cell viability and apoptosis of a breast cancer cell line, MCF-7, induced through the mitochondrial pathway. The result showed that HSF decreased cell viability in MCF-7 cells in a dose- and time-dependent manner, resulting in an increase in the sub-G1 phase cell population. HSF increased the level of the pro-apoptotic Bax protein and decreased the levels of the anti-apoptotic Bcl-2 protein. In addition, HSF induced apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytochtome c, and cleavage of PARP. Taken together, these findings suggest that HSF inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway.

• 한국산학기술학회논문지
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• 제10권3호
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• pp.648-653
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• 2009

p62는 산화스트레스가 주요 원인 중 하나인 퇴행성 뇌질환과 연관된 엉긴 물질(aggregate)의 주요 구성성분이다. 퇴행성 뇌질환과 관련된 것으로 알려진 hydroxydopamin이나 $C_2-ceramide$를 신경모세포종 세포주인 SH-SY5Y에 처리하였을 때 p62의 발현이 유도되며 시간이 지날수록 그 양이 증가되었다. 또한 p62를 과발현시키면 ceramide에 의한 SH-SY5Y 세포의 사멸이 지연되었다. 이 과정에서 P62는 시간이 경과됨에 따라 침전화되며 절단되었다. 이 결과로 p62의 신경보호효과와 여러 종류의 퇴행성 뇌질환에서 p62가 엉긴 물질에서 발견되는 이유를 추측할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.