• KSBB Journal
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• v.16 no.6
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• pp.562-567
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• 2001

Aspergillus niger LK harboring the enantioselective epoxide hydrolase (EHase) activity was isolated, and enantioselectivity of EHase was tested for various racemic aromatic epoxides. The gene encoding epoxide hydrolase was cloned from cDNA library generated by reverse transcriptase-polymerase chain reaction of the isolated total mRNA. Sequence analysis showed that the cloned gene encodes 398 amino acids with a deduced molecular mass of 44.5 kDa. Database comparison of the amino acid sequence reveals that it is similar to fungal EHase, whereas the sequence identity with bacterial EHase is very low. Recombinant expression of the cloned EHase in Escherichia coli BL21 yielded an active EHases, which can offer a potential biocatalyst for the production of chiral epoxides.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1551-1556
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• 2014

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.

• Development and Reproduction
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• v.19 no.3
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• pp.135-144
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• 2015

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

• Journal of Dairy Science and Biotechnology
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• v.33 no.1
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• pp.83-91
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• 2015

Yogurt is a product of the acidic fermentation of milk, which affects the survival of lactic acid bacteria (LAB). The aim of this present study was to examine the survival and acid stress response of Lactobacillus rhamnosus GG to low pH environment. The survival of LAB in commercial yogurt was measured during long-term storage. The enumeration of viable cells of LAB was determined at 15-day intervals over 52-weeks at $5^{\circ}C$. L. acidophilus, L. casei, and Bifidobacterium spp. showed low viability. However, L. rhamnosus GG exhibited excellent survival throughout the refrigerated storage period. At the end of 52-weeks, L. rhamnosus GG survived 7.0 log10 CFU/mL. $F_0F_1$ ATPase activity in L. rhamnosus GG at pH 4.5 was also evaluated. The ATPase activities of the membranes were higher when exposed at pH 4.5 for 24 h. The survival of L. rhamnosus GG was attributable to the induction in $F_0F_1$ ATPase activity. In addition, the mRNA expression levels of acid stress-inducible genes at low pH were investigated by qRT-PCR. clpC and clpE genes were up-regulated after 1 h, and atpA and dnaK genes were up-regulated after 24 h of incubation at pH 4.5. These genes could enhance the survival of L. rhamnosus GG in the acidic condition. Thus, the modulation of the enzymes or genes to assist the viability of LAB in the low pH environment is thought to be important.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.1
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• pp.323-333
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• 2016

Background: Ovarian cancer remains a major worldwide health care issue due to the lack of satisfactory diagnostic methods for early detection of the disease. Prior studies on the role of serum cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) in detecting ovarian cancer presented conflicting results. New tools to improve the accuracy of identifying malignancy are urgently needed. We here aimed to evaluate the diagnostic utility of tissue CA125 and HE4 gene expression in comparison to serum CA125 and HE4 in discriminating benign from malignant pelvic masses. Materials and Methods: One-hundred Egyptian women were enrolled in this study, including 60 epithelial ovarian cancer (EOC) patients and 20 benign ovarian tumor patients, as well as 20 apparently healthy women. Preoperative serum levels of CA125 and HE4 were measured by immunoassays. Tissue expression levels of genes encoding CA125 and HE4 were determined by quantitative real time polymerase chain reaction (qRT-PCR). The diagnostic performance of CA125 and HE4, measured either as mRNA or protein levels, was evaluated by receiver operating characteristic (ROC) curves. Results: The serum CA125+HE4 combination and serum HE4, with area under the curve (AUC) values of 0.935 and 0.932, respectively, performed significantly better than serum CA125 (AUC=0.592; P<0.001). Tissue CA125 and HE4 (AUC=1) performed significantly better than serum CA125 (P<0.001), serum HE4 (P=0.016) and the serum CA125+HE4 combination (P=0.018). Conclusions: Measurement of tissue CA125 and HE4 gene expression not only improves discriminatory performance, but also broadens the range of differential diagnostic possibilities in distinguishing EOC from benign ovarian tumors.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.170-170
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• 2002

College of Pharmacy, Ewha womans University, Seoul, 120-750, Korea 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent halogenated aromatic hydrocarbon congener that induces expression of several genes including CYP1A1. Exposure to TCDD results in many toxic actions such as carcinogenesis, hepatotoxicity, immune suppression, and reproductive and developmental toxicity. Dramatic differences in dioxin toxicity have been observed between the sexes of some animal species, suggesting hormonal modulation of dioxin action. Many studies have been reported and propose several mechanisms of anti-estrogenic effects of TCDD. In contrast, the effect of estrogen on the regulation of CYP1A1 are not clear at present. There are several reports showing conflicting results. It seems that induction/inhibition of CYP1A1 may be dependent on cell-type and concentration. The purpose of this study was to investigate the regulation of TCDD-induced CYP1A1 gene expression by estradiol and its metabolites. We examined whether estradiol and its metabolites altered TCDD-mediated induction of CYP1A1 enzyme activity. 17 ${\beta}$ estradiol and 16 ${\alpha}$ estriol at non cytotoxic concentrations caused a significant concentration dependent decline of TCDD-induced EROD activity To determine whether reduced EROD activity reflected altered CYP1A1 mRNA expression, we measured CYP1A1 mRNA level by RT-PCR. And to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level, we also peformed transient transfection with an AhR responsive reporter plasmid containing the 5' flanking region of the human CYP1A1 gene to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.347-351
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• 2002

Transgenes in HSY-TK gene driven by the lck promoter was tested for the expression in immune cells (Jurkat cells) to apply xenotransplantation of human cells into transgenic animals for the potential use of the proliferation or differentiation of human stem cells in the large animal such as an pig. Also, lck-CFP gene was used for transfection experiment into Jurkat cell to confirm the proper regulation of lck promoter for transgene expression in the T cells. Transfection of lck-GFP gene into Jurkat ceils induced CFP expression in transfected cells. The expression of Ick-TK and Ick-CFP genes was confirmed by RT-PCR using RNAs extracted from Jurkat cells, When Jurkat cells transfected with TK and CFP genes were selected against G418 or gancyclovir treatments, Jurkat cells transfected with TK gene were not proliferated in G4i8 and gancyclovir medium while intact cells or cells transfected with CFP gene could grow in gancyclovir medium. However, Jurkat cells transfected with TK or GFP gene were proliferated in G418 medium probably due to Neo$^{r}$ gene in the vector. Gancyclovir treatment destroyed Jurkat cells expressing TK gene indicating that T-cells expressing TK gene can be selectively eliminated by TK gene expression driven by lck promoter.

• Biomolecules & Therapeutics
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• v.8 no.2
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• The Plant Pathology Journal
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• v.32 no.1
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• pp.25-32
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• 2016

Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.2
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• pp.414-424
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• 2007

Adenophorae Radix (AR), Liriopis Tuber (LT), Dendrobii Monile (DM), Polygonati Officinalis (PO), Polygonati Rhizoma (PR) have been used to treated a variety condition/diseases in traditional oriental medicine. The present study was conducted to investigate the immunmodulatory effects of the water-extracted AR, LT, DH, PO and PR. In spleen cell proliferation assay, DH was significantly enhanced mitogenic activity compared with control group. In RT-PCR, DH ad PO induced IL-2 and IFNr cytokine gene expression in mouse spleen cells. Methotrexate(MTX), immune supression agent, was significantly inhibited mouse spleen cell proliferation(1600 mg/ml). In spite of MTX treatement, DH and PO sustained the spleen cell proliferation, In the flow cytometry analysis, DH stimulated mouse spleen cells showed an increase in B-cell phenotype (CD45R/B220). The water-extracted DH and PO inhibited NO production and iNOS expression in LPS-stimulated RAW 264.7 macrophage cell. DH induced IL-2 and IFNg gene expression in human peripheral mononuclear cells. The GC-MS analysis show that the main component of water-extracted DH was b-Nitroethyl alcohol. The main components of water-extracted PO were Dipirartril-tropico, Methyl sulfoxide and Demsodrox. These data suggest that among these extracts, DH has a protective effcet of immune suppression caused by MTX. DH may be enhance cellular and humoral immune response by the regulation of cytokine gene expression, NO production and B cell production.