• 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.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• Journal of Life Science
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• v.22 no.9
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• pp.1243-1253
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• 2012

The production and secretion of growth hormone (GH) in the anterior pituitary gland can be induced by several natural products to control cell proliferation, differentiation, and migration. To investigate whether Chungkookjang (CKJ) produced by the fermentation process affects GH-related metabolism, the secretion and the response of GH were observed in pituitary cells and GH target cells. Among six CKJs manufactured by different strains of glycine max, only three CKJs, including Daewon (DW), Daepung (DP), and Taegwang (TG), induced GH secretion from GH3 cells at 5.0 mg/ml concentration. There were no significant changes detected in the viability of any of the cells treated with these CKJs. In addition, the increase in GH secretion from the GH3 cells was dependent on the concentration of the three types of CKJs. The proliferation of cell lines, including MG63 and HepG2 cells, that originated from those derived from the GH target organs was significantly activated by treatment with the GH-containing conditional medium (GCM) harvested from the three CKJ-treated GH3 cells, although their induction rate was different from each other. In these cells, p-STAT5 was maximally translocated into the nucleus of MG63 cells 30 min after DW treatment, while it was translocated in HepG2 cells at 60 min. These results suggest that these three types of CKJ could enhance the secretion of GH, as well as the GCM-derived response, in the two target organs.

• Journal of Life Science
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• v.22 no.9
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• pp.1152-1158
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• 2012

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present paper, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene ($D{\times}5$) from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation method. Two expression cassettes comprised of separate DNA fragments containing only the $D{\times}5$ and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring $D{\times}5$ or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with $D{\times}5$ gene and EHA105 with HPTII gene expressing cassette. Then, among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted with both the $D{\times}5$ and HPTII genes into the rice genome. We reconfirmed integration of the $D{\times}5$ and HPTII genes into the rice genome by Southern blot analysis. Wheat $D{\times}5$ transcripts in $T_1$ rice seeds were examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only the $D{\times}5$ gene were successfully screened at the $T_1$ generation. These results show that a co-infection system with two expression cassettes could be an efficient strategy to generate marker-free transgenic rice plants.

• Journal of Life Science
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• v.22 no.9
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• pp.1224-1230
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• 2012

Cancer chemotherapy drugs command a large share of the market, and the development of new therapeutics with high efficacy and specificity is an active area of study. Recently, the development of cancer therapeutics from natural products targeting angiogenesis has drawn attention due to conventional chemotherapeutics showing serious side effects and resistance in cancer cells. In this study, we investigated the pharmacological efficacy of Gomisin A, an active ingredient of Schizandra chinensis baillon, on tumor growth and metastasis. Administration of Gomisin A at 10 and 100 ${\mu}g/ml$ reduced tumor growth in vivo by $80.5{\pm}8.1%$ and $96.2{\pm}2%$, respectively, compared with positive tumor controls. Treatment of Gomisin A in normal and various tumor cell lines did not exert significant toxicity. Mice treated with Gomisin A at a concentration of 10 and 100 ${\mu}g$/head showed a significant reduction in tumor-induced angiogenesis of $151{\pm}16.9%$ and $98.5{\pm}29.5%$, respectively. Furthermore, tumor metastasis analysis revealed that the administration of Gomisin A at a concentration of 10 and 100 ${\mu}g$/head inhibited tumor metastasis by $13.5{\pm}8.56%$ and $58.3{\pm}9.12%$, respectively. In addition, Gomisin A significantly decreased cell adhesion of the B16BL6 cells to the extracellular matrix. These results demonstrate that Gomisin A inhibits tumor growth via suppression of angiogenesis and tumor metastasis inhibition, without cellular toxicity. The pharmacological efficacy of Gomisin A suggests that it may be a potential candidate for the development of cancer drugs.

• 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 the Korean Geographical Society
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• v.28 no.4
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• pp.285-297
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• 1993

For the application of the diffusion equation, slope height and maximum slope angle are calculated from the plotted slope profile. Using denudation rate as a solution for the diffusion equation, an apparent age index can be calculated, which is the total amount of denudation through total time. Plots of slope angle versus slope height and apparent age index versus slope height are useful for determining relative or absolute ages and denudation rates. Mathematical simulation plots of slope angle versus slope height can generate equal denudation-rate lines for a given age. Mathematical simulations of slope angle versus age for a given slope height, for equal denudation-rate at a particular profile site, and for comparing to other sites having controlled ages.

• KSBB Journal
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• v.19 no.6 s.89
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• pp.471-477
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• 2004

Oldenlandia diffusa is a Chinese medicinal herb with antitumor activity capable of suppressing the growth of some cancer cell lines. Oleanolic acid and ursolic acid are triterpenoid compounds that exist in Oldenlandia diffusa. Recently, these have been noted for anti-inflammatory, anti-cancer, and hepato-protective effects. Application of both plant growth regulators, 2,4-D and kinetin, was found to be essential for the initiation of callus and suspension cells. Leaf blades of Oldenlandia diffusa was transformed into callus on Schenk and Hildebrandt medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L kinetin, while optimum initiation condition for suspension cells of Oldenlandia diffusa was determined to be 0.75 mg/L 2,4-D and 0.1 mg/L kinetin. Chromatographic separation of oleanolic acid from its derivatives was achieved using Rexchrom S5-100-ODS column. Analytical conditions for oleanolic acid were determined as follows: flow rate at 1.0 mL/min, UV length at 200 nm and mobile phase of $80\%$ acetonitrile and $20\%$ water. Production of secondary metabolites was found to be increased by the treatment with elicitors or signal transducers. The maximum production of oleanolic acid was 99.6 mg/L in cultures with 0.5 mM salicylic acid. It is 1.74 times higher than that of control.

• Tuberculosis and Respiratory Diseases
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• v.54 no.5
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• pp.542-550
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• 2003

Background : Synthetic glucocorticoids are widely used in many chronic inflammatory diseases because of their excellent anti-inflammatory activity. Enhancing the transcription of $I{\kappa}B$ and preventing activated NF-${\kappa}B$ from binding to ${\kappa}B$ sites are thought to be the underlying mechanisms. But these data are largely derived from in vitro studies using cell lines. In this study, after administrating a steroid to volunteers, we evaluated the effect on the NF-${\kappa}B$ system. Methods : Prednisolone(0.5mg/kg/d) was orally administered to 5 healthy volunteers for 7 days. Before and after the administration, we sampled their peripheral blood monocytes, and performed western blot analysis both with stimulation, using IL-$1{\beta}$, LPS, TNF, and without stimulation(baseline). We also performed EMSA after stimulation with LPS. Results : After ingestion of the steroid, baseline expressions of $I{\kappa}B{\alpha}$ were increased in two of the subjects, while suppressed degradations of $I{\kappa}B{\alpha}$ to stimulations were observed in all five. In addition, the binding capacity of NF-${\kappa}B$ after the administration was decreased. Conclusion : Steroid plays such roles as enhancing the transcription of $I{\kappa}B{\alpha}$, suppressing the DNA binding capacity of NF-${\kappa}B$, and suppressing the degradation of $I{\kappa}B{\alpha}$.

• Journal of Genetic Medicine
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• v.7 no.1
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• pp.82-86
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• 2010

Jumping translocations (JT) are chromosomal rearrangements involving one donor chromosome and several recipient chromosomes. While JTs are frequently observed as acquired chromosomal abnormalities in hematologic malignancies, constitutional JTs are only rarely reported. We report two cases of constitutional JT in chorionic villi derived from the products of conception. The karyotype of the first case was 46,XY,add(18)(p11.1)[61]/45,XY,der(18;21)(q10;q10)[32]/46,XY,-18,+mar[16]/46,XY,i(18)(q10)[9]/45,XY,der(15;18)(q10;q10)[6]/46,XY,+1,dic(1;18)(p22;p11.1)[2]/45,XY,der(13;18)(q10;q10)[1]/46,XY[32]. The donor was a chromosome 18. The recipient chromosomes were chromosomes 1, 13, 15, 18 and 21. In the second case, the karyotype was 46,XY,der(22)t(9;22)(q12;q13)[22]/46,XY,der(22)t(1;22)(q21;q13)[13]/46,XY,add(22)(q13)[5]/46 XY[23]. The donor was a chromosome 22 and recipients were chromosomes 1 and 9. Both cases were de novo. The breakpoints of chromosomes were mostly in centromeric regions, pericentromeric regions, or telomeric regions. Normal cell lines were observed in both cases. This report supports the prior findings that the unstable nature of JT, resulting in chromosomal imbalance, most likely contributed to these early miscarriages.