• Korean Journal of Food Science and Technology
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• v.44 no.1
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• pp.55-60
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• 2012

The aim of this study was to determine the optimal physical treatment to reduce the antigenicity of gliadin in wheat dough. Medium wheat dough was treated with an autoclave (5, 10, 30, and 50 min at $121^{\circ}C$, 1 atm), a microwave (1, 5, and 10 min) or both (10, 30, and 50 min/5, 10 min). The proteins in the dough extracts were analyzed by SDSPAGE and the binding ability of anti-gliadin IgG to gliadin was examined by ci-ELISA and immunoblotting. Results showed that the ability of anti-gliadin IgG to bind to gliadin in wheat dough treated with an autoclave alone or in combination with a microwave was decreased. Especially, it declined to ~77% after autoclaving for 30 min and 35% after both autoclaving for 50 min and microwaving for 5 min. In addition, the intensity of gliadin bands in SDS-PAGE were weakened and anti-gliadin IgG did not recognize gliadin in immunoblotting. However, microwaving alone did not affect the antigenicity of gliadin in wheat dough. These results indicate that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat dough. Moreover, autoclaving in combination with microwaving is more effective for reducing the antigenicity of wheat dough.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• Journal of Life Science
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• v.26 no.6
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• pp.663-672
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• 2016

The Cnidium monnieri (L.) Cusson is an annual plant distributed in China and Korea. The fruit of C. monnieri is used as a medicinal herb that is effective for the treatment of carbuncle and pain in female genitalia. However, the anti-cancer effects of CME have not yet been reported. In this study, we assessed the apoptotic effects and cell cycle arrest effects of ethanol extracts from C. monnieri on HCT116 colon cancer cells. The results of an MTT assay and LDH assay demonstrated a decrease in cell viability and the cytotoxic effects of CME. In addition, the number of apoptotic body and the apoptotic rate were increased in a dose-dependent manner through Hoechst 33342 staining and Annexin V-PI double staining. In addition, cell cycle arrest occurred at the G1 phase by CME. Protein kinase B (Akt) plays an important role in cancer cell survival, growth, and division. Akt down-regulates apoptosis-mediated proteins, such as mammalian target of rapamycin (mTOR), p53, and Glycogen Synthase kinase-3β (GSK-3β). CME could regulate the expression levels of p-Akt, p-mTOR, p-GSK-3β, Bcl-2 family members, caspase-3, and PARP. Furthermore, treatment with CME, LY294002 (PI3K/Akt inhibitor), BIO (GSK-3β inhibitor), and Rapamycin (mTOR inhibitor) showed that apoptotic effects occurred through the regulation of the AKT/mTOR/GSK-3β signaling pathway. Our results demonstrated CME could induce apoptosis and cell cycle arrest in HCT116 colon cancer cells.

• Journal of Life Science
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• v.26 no.6
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• pp.689-697
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• 2016

Kefir is an acidic-alcoholic fermented milk product originating from the Caucasian mountains. Kefir has long been known for its probiotic health benefits, including its immunomodulatory effects. The objectives of this study were to investigate the properties of a fermented whey product and to examine the effects of kefir grains on the in vitro immune-modulation of human mast cell-1 (HMC-1). The results showed that the whey fermented by kefir grains contained the maximum lactic acid bacteria and yeast for 16 hr by 1.83×10⁸ and 6.5×10⁵ CFU/ml, respectively, and lactose and whey proteins were partially hydrolyzed. The experimental whey fermented by kefir grains exhibited an in vitro anti-inflammatory effect on the HMC-1 line for 8, 16, and 24 hr, and this effect induced the expression of interleukin (IL)-4 as a pro-inflammatory cytokine, but not for 48 hr by RT-PCR in HMC-1 cells. In addition, the same phenomenon was observed for the expression of IL-8 as a pro-inflammatory cytokine by the kefir-fermented whey during the same periods of 8-48 hr under the same conditions. These cytokines resulted in the production of IL-4 at 20-25 ng in HMC-1 cells for 8, 16, and 24 hr, whereas 5 ng was produced for 48 hr by the fermented whey. In contrast, IL-8 was produced at 15-20 ng in HMC-1 cells during 4, 8, 16, and 24 hr, while 7 ng was produced at 48 hr. It was concluded that the whey fermented by kefir grains possesses a potential anti-inflammatory function, which could be used for an industrial application as an ingredient of functional foods and pharmaceutical products.

• Journal of Life Science
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• v.17 no.10
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• pp.1447-1451
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• 2007

Through the screening of marine natural compounds that inhibit cancer cell proliferation, we previously reported that pectenotoxin-2 (PTX-2) isolated from marine sponges exhibits selective cytotoxicity against several cell lines in p53-deficient tumor cells compared to those with functional p53. However, the molecular mechanisms of its anti-proliferative action on malignant cell growth are not completely known. To further explore the mechanisms of its anti-cancer activity and to test whether the status of p53 in liver cancer cells correlates with their chemo-sensitivities to PTX-2, we used two well-known hepatocarcinoma cell lines, p53-deficient Hep3B and p53-wild type HepG2. We have demonstrated that PTX-2 markedly inhibits Hep3B cell growth and induces apoptosis whereas HepG2 cells are much more resistant to PTX-2 suggesting that PTX-2 seems to act by p53-independent cytotoxic mechanism. The apoptosis induced by PTX-2 in Hep3B cells was associated with the modulation of DNA fragmentation factor (DFF) family proteins, up-regulation of pro-apoptotic Bcl-2 family members such as Bax and Bcl-xS and activation of caspases (caspase-3, -8 and -9). Blockade of the caspase-3 activity by caspase-3 inhibitor, z-DEVD-fmk, prevented the PTX-2-induced growth inhibition in Hep3B cells. Moreover, treatment with PTX-2 also induced phosphorylation of AKT and extracellular-signal regulating kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MARK). Specific inhibitors of PI3K inhibitor (LY294002) and ERK1/2 inhibitor (PD98059) significantly blocks PTX-2-induced-anti-proliferative effects, whereas a JNK inhibitor (SP600125) and a p38 MAPK inhibitor (SB203580) have no significant effects demonstrating that the pro-apoptotic effect of PTX-2 mediated through activation of AKT and ERK signal pathway in Hep3B cells.

• Journal of Life Science
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• v.21 no.1
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• pp.44-55
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• 2011

This study aimed to investigate the effects of water extract of Rubus coreanus (RCE) on the expression and activity of endothelial nitric oxide synthase (eNOS), as well as its signal transduction pathways in human umbilical vein endothelial cells (HUVECs). The specific inhibitors of NOS show RCE treatment increases NO production in HUVECs due to the up-regulation of eNOS rather than iNOS. The real-time expression level of eNOS mRNA was also increased upon RCE treatment in HUVECs. While a PKC-specific inhibitor, RO-317549, did not alter RCE-induced NO production in HUVECs, tamoxifen (estrogen receptor-specific inhibitor), PD98059 (ERK-specific inhibitor) and LY-294002 (PI3K/Akt-specific inhibitor) did have suppressive effects. Increased NO production by RCE seems to result from a higher level of active eNOS (pSer1177). Specifically, inhibition of ERK not only decreased the level of active eNOS, but also increased the inactive form of the enzyme (pThr495) in HUVECs. This study suggests that RCE treatment increases NO production in HUVECs due to the increased expression and activity of eNOS. It is also shown that RCE-induced eNOS activation occurs partly through the binding of RCE to the estrogen receptor, along with ERK and PI3K/Akt-dependent signal transduction pathways. In addition, the regulatory binding proteins of eNOS including Hsp90 and caveolin-1 were related to these effects of RCE on eNOS activity in HUVECs.

• Journal of Life Science
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• v.21 no.1
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• pp.146-154
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• 2011

In this study, physiological changes in a thermotolerant yeast Saccharomyces cerevisiae KNU5377 cell exposed to 48-hour alcohol fermentation at $40^{\circ}C$ were investigated. After 12 hours of alcohol fermentation at $40^{\circ}C$, the $C_{16:1}$ unsaturated acid of plasma membrane increased to 1.5 times more than the $C_{16:0}$ saturated fatty acid, and to about 2 times more for the $C_{18:1}$ unsaturated fatty acid. Fermentation at both $30^{\circ}C$ and $37^{\circ}C$ fermentation showed the same pattern as that done at $40^{\circ}C$. The pH of the alcohol-fermentation medium was reduced to pH 4.1 from a starting pH of 6.0 through the 12-hr fermentation and then maintained this level during the continuing fermentation. With the process of fermentation, the remaining glucose was reduced, but its amount remaining during the $40^{\circ}C$-fermentation was less reduced than those fermented at $30^{\circ}C$ and $37^{\circ}C$. In the study investigating the changing pattern of cellular proteins in the alcohol-fermenting cells, the SDS-PAGE and 2-D data indicated the most expressed dot was phosphoglycerate kinase, which is one enzyme involved in glycolysis. Why this enzyme was most expressed in the cells exposed to unfavorable conditions such as high temperature, increasing concentration of produced alcohol and long time exposure to other stress factors remains unsolved.

• Journal of Life Science
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• v.22 no.10
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• pp.1359-1370
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• 2012

Arabidopsis AtERF71/HRE2, a transcription activator, is located in the nucleus and is involved in the signal transduction of low oxygen and osmotic stresses. In this study, microarray analysis using AtERF71/HRE2-overexpressing transgenic plants was performed to identify genes downstream of AtERF71/HRE2. A total of 161 different genes as well as AtERF71/HRE2 showed more than a twofold higher expression in AtERF71/HRE2-overexpressing transgenic plants compared with wild-type plants. Among the 161 genes, 24 genes were transcriptional regulators, such as transcription factors and DNA-binding proteins, based on gene ontology annotations, suggesting that AtERF71/HRE2 is an upstream transcription factor that regulates the activities of various downstream genes via these transcription regulators. RT-PCR analysis of 15 genes selected out of the 161 genes showed higher expression in AtERF71/HRE2-overexpressing transgenic plants, validating the microarray data. On the basis of Genevestigator database analysis, 51 genes among the 161 genes were highly expressed under low oxygen and/or osmotic stresses. RT-PCR analysis showed that the expression levels of three genes among the selected 15 genes increased under low oxygen stress and another three genes increased under high salt stress, suggesting that these genes might be downstream genes of AtERF71/HRE2 in low oxygen or high salt stress signal transduction. Microarray analysis results indicated that AtERF71/HRE2 might also be involved in the responses to other abiotic stresses and also in the regulation of plant developmental processes.

• Journal of Life Science
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• v.24 no.6
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• pp.618-625
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• 2014

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation. Two expression cassettes, consisting of separate DNA fragments containing Glu-1Dy10 and hygromycin phosphotransferase II (HPTII) resistance genes, were introduced separately into Agrobacterium tumefaciens EHA105 for co-infection. Each EHA105 strain harboring Glu-1Dy10 or HPTII was infected into rice calli at a 3: 1 ratio of Glu-1Bx7 and HPTII. Among 290 hygromycin-resistant $T_0$ plants, we obtained 29 transgenic lines with both the Glu-1Dy10 and HPTII genes inserted into the rice genome. We reconfirmed the integration of the Glu-1Dy10 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the Glu-1Dy10 in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the Glu-1Dy10 gene were successfully screened in the $T_1$ generation.

• Journal of Life Science
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• v.27 no.9
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• pp.1070-1077
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• 2017

Chondrocyte apoptosis induced by reactive oxygen species (ROS) plays an important role in the pathogenesis of osteoarthritis. Schisandrin A, a bioactive compound found in fruits of the Schisandra genus, has been reported to possess multiple pharmacological and therapeutic properties. Although several studies have described the antioxidant effects of analogues of schisandrin A, the underlying molecular mechanisms of this bioactive compound remain largely unresolved. The present study investigated the cytoprotective effect of schisandrin A against oxidative stress (hydrogen peroxide [$H_2O_2$]) in SW1353 human chondrocyte cells. The results showed that schisandrin A preconditioning significantly inhibited $H_2O_2-induced$ growth inhibition and apoptotic cell death by blocking the degradation of poly (ADP-ribose) polymerase proteins and down-regulating pro-caspase-3. These antiapoptotic effects of schisandrin A were associated with attenuation of mitochondrial dysfunction and normalization of expression changes of proapoptotic Bax and antiapoptotic Bcl-2 in $H_2O_2-stimulated$ SW1353 chondrocytes. Furthermore, schisandrin A effectively abrogated $H_2O_2-induced$ intracellular ROS accumulation and phosphorylation of histone H2AX at serine 139, a widely used marker of DNA damage. Thus, the present study demonstrates that schisandrin A provides protection against $H_2O_2-induced$ apoptosis and DNA damage in SW1353 chondrocytes, possibly by prevention of ROS generation. Collectively, our data indicate that schisandrin A has therapeutic potential in the treatment of oxidative disorders caused by overproduction of ROS.