• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1777-1784
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• 2020

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

• 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.

• Oncology Letters
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• v.18 no.3
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• pp.3256-3264
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• 2019

The induction of apoptosis is a useful strategy in anti-cancer research. Various Moon Hyung Yang (MHY) compounds have been developed as novel anti-cancer drug candidates; in the present study, the pro-apoptotic effects of (Z)-5-(3-ethoxy-4-hydroxybenzylidene)-2-thioxothiazolidin-4-one (MHY695) on HCT116 human colon cancer cells were assessed. MTT assays were performed to investigate the dose-dependent cytotoxic effects of MHY695 on HCT116 cells. Immunofluorescence staining and flow cytometry analyses were performed to identify apoptotic cell death, and western blot analysis was used to investigate the apoptotic-signaling pathways. A mouse xenograft model was also used to determine the effects of MHY695 in vivo. MHY695 decreased the viability of HCT116 cells and induced apoptotic cytotoxicity. The apoptotic mechanisms induced by MHY695 involved the dephosphorylation of Bcl-2-associated agonist of cell death protein following protein kinase B inactivation, induced myeloid leukaemia cell differentiation protein and BH3-interacting domain death agonist truncation, caspase-3 and -9 activation and poly (ADP-ribose) polymerase cleavage. In addition, MHY695 significantly suppressed tumor growth in the mouse xenograft model, compared with the vehicle control. Notably, MHY695 exhibited potent anti-cancer effects in four different types of human colon cancer cell line, including Caco-2, DLD-1, HT-29 and HCT116. Additionally, MHY695 showed reduced cytotoxicity in NCM460, normal colonic epithelial cells. Furthermore, MHY-induced cytotoxicity in colon cancer cells was independent of the tumor suppressor protein p53. Collectively, these observations suggested that MHY695 may be a novel drug for the treatment of colon cancer.

• Journal of Life Science
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• v.25 no.2
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• pp.249-255
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• 2015

Schisandrae fructus [Schizandra chinensis (Turcz.) Baillon] is a medicinal herb widely used for treating various inflammatory and immune diseases in East Asian countries. The Schisandrae Semen essential oil (SSeo) from this plant has pharmacological activities, including antioxidant, antimicrobial, and antitumoral activities. Nevertheless, the biological activities and underlying molecular mechanisms of the potential anti-cancer effects of this oil remain unclear. In the present study, we investigated the potential inhibition of apoptosis signaling pathways by SSeo in human leukemia U937 cells and evaluated the underlying molecular mechanism. Exposure to SSeo resulted in a concentration-dependent growth inhibition due to apoptosis, which was verified by DNA fragmentation, the presence of apoptotic bodies, and an increase in the sub-G1 ratio. Induction of apoptotic cell death by SSeo was correlated with the down-regulation of members of the inhibitor of apoptosis protein (IAP) family (including X-linked inhibitor of apoptosis protein (XIAP), cIAP-1, and surviving) and anti-apoptotic Bcl-2, and with up-regulation of death receptor (DR) 4 and DR5, depending on dosage. SSeo treatment also induced Bid truncation, mitochondrial dysfunction, proteolytic activation of caspase-3, -8 and -9, and concomitant degradation of activated caspase-3 target proteins such as poly (ADP-ribose) polymerase. Taken together, these findings suggest that SSeo may be a potential chemotherapeutic agent for use in the control of human leukemia cells. Further studies are needed to identify its active compounds.

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.273-282
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• 2000

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-l-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of ${\Delta}rmlC$ E. coli strain $S{\Phi}874$ (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.239-244
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• 2014

ErmSF is one of the four antibiotic resistance factor proteins expressed by Streptomyces fradiae, antibiotic tylosin producer, which renders $MLS_B$ (macrolide-lincosamide-streptogramin B) antibiotic resistance through dimethylating A2058 of 23S rRNA, thereby reducing the affinity of antibiotic to ribosome. Unlike other Erm proteins, ErmSF harbors long N-terminal end region. To investigate its role in enzyme activity, mutant ErmSF deleted of 1-38 amino acids was overexpressed and activity in vivo and in vitro was observed. In vitro enzymatic assay showed that mutant protein exhibited reduced activity by 20% compared to the wild type enzyme. Due to the reduced activity of the mutant protein, cells expressing mutant protein showed weaker resistance to erythromycin than cells with wild type enzyme. Presumably, the decrease in enzyme activity was caused by the hindrance in substrate binding and (or) product release, not by defect in the methyl group transfer occurred in active site.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5871-5874
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• 2012

Around 1.35 million people of worldwide suffer from breast cancer each year, whereas in India, 1 in every 17 women develops the disease. Mutations of the Breast Cancer 1 (BRCA1) gene account for the majority of breast/ovarian cancer families. The purpose of study was to provide a prevalence of BRCA1 germline mutations in the North-East Indian population. In relation to the personal and family history with the breast cancer, we found mutations in 6.25% and 12.5% respectively. Three mutations, 185DelAG, 1014DelGT and 3889DelAG, were observed in our North-East Indian patients in exons 2 and 11, resulting in truncation of the BRCA1 protein by forming stop codons individually at amino acid positions 39, 303 and 1265. Our results point to a necessity for an extensive mutation screening study of high risk breast cancer cases in our North-East Indian population, which will provide better decisive medical and surgical preventive options.

• Toxicological Research
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• v.30 no.1
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• pp.33-38
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• 2014

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a $Ni^{2+}$-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.3
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• pp.343-348
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• 2018

Recent human genetic studies have shown that $G{\beta}5$ is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of $G{\beta}5$ in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of $G{\beta}5$. The cells expressing $G{\beta}5$ had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. $G{\beta}5$ also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of $G{\beta}5$-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.

• BMB Reports
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• v.52 no.5
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• pp.330-335
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• 2019

Hepatitis B virus (HBV) encoding the HBV x protein (HBx) is a known causative agent of hepatocellular carcinoma (HCC). Its pathogenic activities in HCC include interference with several signaling pathways associated with cell proliferation and apoptosis. Mutant C-terminal-truncated HBx isoforms are frequently found in human HCC and have been shown to enhance proliferation and invasiveness leading to HCC malignancy. We investigated the molecular mechanism of the reduced doxorubicin cytotoxicity by C-terminal truncated HBx. Cells transfected with C-terminal truncated HBx exhibited reduced cytotoxicity to doxorubicin compared to those transfected with full-length HBx. The doxorubicin resistance of cells expressing C-terminal truncated HBx correlated with upregulation of the ATP binding cassette subfamily B member 1(ABCB1) transporter, resulting in the enhanced efflux of doxorubicin. Inhibiting the activity of ABCB1 and silencing ABCB1 expression by small interfering ribonucleic acid (siRNA) increased the cytotoxicity of doxorubicin. These results indicate that elevated ABCB1 expression induced by C-terminal truncation of HBx was responsible for doxorubicin resistance in HCC. Hence, co-treatment with an ABCB1 inhibitor and an anticancer agent may be effective for the treatment of patients with liver cancer containing the C-terminal truncated HBx.