• Journal of Life Science
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• v.10 no.2
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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.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.888-891
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• 2008

Twenty-three fungal strains were isolated from meju that had originated from the Sunchang province, the famous location for making fermented soybean foods in Korea. The restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rDNA (ITS-RFLP) was applied to differentiate the isolated fungal strains. First, the ITS region by polymerase chain reaction (PCR) with specific primers was amplified and then cleaved the products with different restriction enzymes. Cleavage of the amplified fragments with the restriction enzymes AluI, HaeIII, HhaI, and TaqI revealed extensive polymorphisms. The ITS-RFLP results highly correlated with ITS sequence analysis. All of the 23 fungal strains were classified into 5 groups by ITS-RFLP analysis. Aspergillus oryzae was the major fungal strain isolated from Sunchang meju (12 out of 23), while Aspergillus fumigatus was the next most frequently isolated strain (7 out of 23). In contrast, it was found that Fusarium asiaticum, Aspergillus sydowii, and Arthrinium sp. were the minor fungal strains in meju.

• Molecules and Cells
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• v.42 no.11
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1343-1349
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• 2021

Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the Periplaneta americana transcriptome and detected periplanetasin-5 using an in silico antimicrobial peptide prediction method. In this study, we investigated whether periplanetasin-5 has anticancer activity against the human leukemia cell line K562. Cell growth and survival of K562 cells treated with periplanetasin-5 were decreased in a dose-dependent manner. By using flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation, we found that periplanetasin-5 induced apoptotic and necrotic cell death in leukemia cells. In addition, these events were associated with increased levels of the pro-apoptotic proteins Fas and cytochrome c and reduced levels of the anti-apoptotic protein Bcl-2. Periplanetasin-5 induces the cleavage of pro-caspase-9, pro-caspase-8, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP). The above data suggest that periplanetasin-5 induces apoptosis via both the intrinsic and extrinsic pathways. Moreover, caspase-related apoptosis was further confirmed by using the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), which reversed the periplanetasin-5-induced reduction in cell viability. In conclusion, periplanetasin-5 caused apoptosis in leukemia cells, suggesting its potential utility as an anticancer therapeutic agent.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.19-19
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• 2001

Mammalian urothelium undergoes unique membrane specialization by making the asymmetric unit membrane (AUM) that is covered with the apical cell surface during terminal differentiation. The AUM contains several major integral membrane proteins including uroplakin Ia, Ib, II and III. The genes for uroplakins have been cloned from humans and mice, but not from porcine. In this study, we report the cloning of the UPII genomic DNA, which codes for the full length open reading frame for the uroplakin II protein. The deduced amino acid sequence encodes of a hydrophobic NH$_2$-terminal peptide, a prosequence, and a mature protein. The prosequence contains three potential N-glycosylation sites and a RGRR cleavage site that may be involved in uroplakin II processing and maturation. Northern and immunohistochemistry analyses showed that the porcine UPII gene is only expressed in urothelium and that the protein was specifically localized in urothelial superficial cells. A 2kb of upstream in the promoter sequence contains multiple transcription factor binding sites, including GC-box, SPI, AP2, and GATA-box sites, but not for TATA or CAAT-box sequences. Comparison of the porcine UPII promoter sequence with that of the murine by MEME system presented two conserved motifs, suggesting a cis-acting regulatory role for the conserved sequences. Sequence homology between two species in motif A and B was 79% and 80% respectively, although their relative locations were different. During the gestation, mouse bladder at estrus stages and day 10 after parturition showed higher UPII expression, while showed lower expression at peri-implantation stage. Taken together, our results showed that the porcine UPII gene was expressed highly and specifically in the bladder urothelium and that steroid hormones for implantation changed the expression of UPII in the bladder, although the biological significance of UPII remains to be not determined.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.60-60
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• 2001

Human thrombopoietin (hTPO) is a cytokine that plays a central role in megakaryopoiesis by influencing on the development and maturation of megakaryocyte and platelet production. To induce hTPO production in the mammary gland, expression vector was constructed by combining the promoter of bovine beta-casein gene, cDNA of hTPO and neomycine resistance gene for transfection into fibroblasts. Bovine fibroblast cells derived from female ear skin were transfercted with the expression vector using Lipofectamine (Life Technology, NY). Transected cells resistant to G4l8 treatment (600 $\mu\textrm{g}$/$m\ell$) were recovered and colony formation was initiated at 13 days. The colonies with about 1 cm diameter were picked and analysed by PCR. Single transfected cells were individually transferred to enucleated oocytes. After electrofusion, the reconstructed embryos were exposed to calcium ionophore (5uM) for 5 min followed by treatment with 6-DMAP (2.5 mM) for 4h. The nuclear transfer embryos were cultured in CRlaa medium at 38.5C, 5% $CO_2$ for 7 days. Twenty three of 29 (79.3%) colonies were proved to be hTPO transfectants by PCR. The colonies were further passaged and used to produce transgenic embryos using nuclear transfer. Cleavage and developmental rates of reconstructed embryos to the blastocyst stage were 65.1% and 39.4%, respectively Of 22 blastocysts that developed from reconstructed embryos with the transfected cell, 20 embryos (90.9%) were positive for hTPO by using PCR analysis. The results suggest that somatic cell nuclear transfer is efficient for production of transgenic embryos.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.77-77
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• 2020

Purple loosestrife-Lythrum anceps (Koehne) Makino is a herbaceous perennial plant belonging to the Lythraceae family. It has been used for centuries in Korea and other Asian traditional medicine. It has been showed pharmacological effects, including anti-oxidant and anti-microbial effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, we investigated the mechanism of apoptosis signaling pathways by ethanol extract of Lythrum anceps (Koehne) Makino (ELM) in human leukemia U937 cells. Treatment with ELM significantly inhibited cell growth in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies (ApoBDs), DNA fragmentation and increased populations of sub-G1 ratio. Induction of apoptosis by ELM was connected with up-regulation of death receptor (DR) 4 and DR5, pro-apoptotic Bax protein expression and down-regulation of anti-apoptotic Bcl-2 protein, and inhibitor of apoptosis protein (IAP) family proteins (XIAP, cIAP-1, survivin), depending on dosage. This induction was associated with Bid truncation, mitochondrial dysfunction, proteolytic activation of caspases (-3, -8 and -9) and cleavage of poly(ADP-ribose) polymerase protein. Therefore, our data indicate that ELM suppresses U937 cell growth by activating the intrinsic and extrinsic apoptosis pathways, and thus may have applications as a potential source for an anti-leukemic chemotherapeutic agent.

• KSBB Journal
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• v.16 no.5
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• pp.486-492
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• 2001

The antimutagenic mechanism of the fraction III(RG III)separated from the water extract of Rehmannia glutionosa was investigated by Escherichia. coli GW and B/r strains. RG-III treatment did not affect the ${\beta}$-galactosidase activity E. coli GW-1060, 1106, 1107 and 1105. These results indicated that RG-III did not induce RecA protein amplification and did not also prevent the proteolytic cleavage of LexA. The bio-antimutagenicity and survival effect of RG-III on 4-nitroquinoline 1-oxide(4NQO), N-methyl-N-nitor-N\`-nitrosoguanidine(MNING) were investigate by E. coli B/r strains with have different pathway of DNA repai. RG-III slightly increased the survival of 4NQO-treated WP2, WP2s, WP67, CM561, CM611 cells, but the reactivation of survival cannot ve explained by the repair mode. RG-III caused the decrease of mutagenicity and lethality treated with MNNG in ZA159 despite of the increase in WP2, WP2s, WP67, CW561, CM611. Compared with bio-antimutagenic effects of RG-III on 4NQO, greatly increased antimutagenic effects of RG-III were observed with all the E. coli B/r strains tested, but less active in ZA159. These results suggest that RG-III was identified as a blocking agent for preventing the 4NQO induced mutagenesis, and may act as chl-products.

• Journal of Life Science
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• v.20 no.12
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• pp.1820-1828
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• 2010

Cobalt(II) chloride, a chemical compound with the formula$CoCl_2$, has been widely used in the treatment of anemia, as a chemical agent for the induction of hypoxia in cell cultures, and is known to activate hypoxic signaling. However, excessive exposure to cobalt is associated with several clinical conditions, including asthma, pneumonia, and hematological abnormalities, and can lead to tissue and cellular toxicity. It is also known to induce apoptosis. One of the questions was that of whether $CoCl_2$ might induce apoptosis via endoplasmic reticulum (ER) stress in neurons. To address this question, first, the level of DNA fragmentation was measured for assay of apoptotic rates using $CoCl_2$ with neuron PC12 cells. After confirmation of apoptosis inductions, under the same conditions, the expression levels of ER stress associated factors [ER chaperones Bip, calnexin, ERp72, ERp29, PDI, and ER membrane kinases (IRE1, ATF6, PERK)] were examined by RT-PCR and Western blotting. These results indicated that apoptosis is induced through activation of ER membrane kinases via ER stress. In conclusion, during induction of apoptosis through $CoCl_2$-induced hypoxia in neuron PC12 cells, ER membrane kinase of IRE1 was dominantly up-expressed, and, consecutively, TRAF2, which has been suggested to be one of the links connecting apoptosis and ER stress, was strongly up-expressed.