• Journal of Life Science
• /
• v.16 no.4
• /
• pp.571-578
• /
• 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
• /
• v.16 no.7 s.80
• /
• pp.1133-1140
• /
• 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
• /
• v.13 no.3
• /
• pp.314-323
• /
• 2003

The purpose of this study was to examine the effects of xenobiotic nuclear receptors, CAR, SXR, and PPAR${\gamma}$ on the transcriptional activity of estrogen receptor in human breast cancer cell lines and compare with those in human hepatoma cell line. Two different breast cancer cell lines, MCF-7 and MDA-MB-231 were cultured and effects of CAR, SXR, and PPAR${\gamma}$ on the ER-mediated transcriptional activation of synthetic (4ERE)-tk-luciferase reporter gene were analyzed. Consistent with the previous report, CAR significantly inhibited ER-mediated transactivation and SXR repressed modestly whereas the PPAR${\gamma}$ did not repress the ER-mediated transactivation. However, in breast cancer cells neither of the xenobiotic receptors repressed the ER-mediated transactivation. Instead, they tend to increase the transactivation depending on the cell type and xenobiotic nuclear receptors. In MCF-7, SXR but neither CAR nor PPAR${\gamma}$ slightly increased ER-mediated transactivation whereas in MDA-MB-231, CAR and PPAR${\gamma}$ but not SXR tend to increase the transactivation of the reporter gene. These results indicate that the effects of ER cross-talk by the CAR, SXR, and PPAR${\gamma}$ , are different in breast cancer cells from hepatoma cells. In conclusion, the transcriptional regulation by estrogen can involve different cross-talk interaction between estrogen receptor and xenobiotic nuclear receptors depending on the estrogen target cells.

• Korean Journal of Acupuncture
• /
• v.17 no.1
• /
• pp.47-65
• /
• 2000

The meridian, the meridian point and the Acupuncture-Bu-Xie(鍼灸補瀉) of oriental medicine are very important in the Department of Acupuncture and Moxibustion. Until now it has been confused at the practical use, and it showed up many transformation to the ages and many scholars. And then, I made a study of effects on the thermal changes of Sangyang($LI_1$,商陽), Igan($LI_2$,二間), Samgan($LI_3$,三間), Hapkok($LI_4$,合谷), Yanggye($LI_5$,陽谿), Kokchi($LI_{11}$,曲池), Yonghyang($LI_{20}$,迎香) following acupuncture on the Hapkok with the Rotated Acupuncture-Bu-Xie(捻轉補瀉) stimulation. This study researched into clinical statistics for 140 men who are in good health, and they are studying oriental medicine at Woosuk university in Korea. This study was covered a period of 3 months form June, 1999 to August, 1999. The objective was divided into seven groups, those are the control group(CON, N=20), the acupuncture stimulation group with non-rotation on Hapkok of left hand(A-I, N=20), the acupuncture stimulation group with non-rotation on non-meridian point(NA) of left hand(A-II, N=20), the acupuncture stimulation group with Bu-rotation(捻轉補法) on Hapkok of left hand(B-I, N=20), the acupuncture stimulation group with Bu-rotation on non-meridian point(NA) of left hand(B-II, N=20), the acupuncture stimulation group with Xie-rotation(捻轉瀉法) on Hapkok of left hand(C-I, N=20), and the acupuncture stimulation group with Xie-rotation on non-meridian point of left hand(C-II, N=20). The first, I took a picture for 140 men with the Digital infrared thermal image(D.I.T.I.). After 10 minutes, I took a second picture for each group following experimental methods, those were followed acupuncture on the Hapkok and the non-meridian point with the retentive and Rotated Acupuncture-Bu-Xie stimulation. The results are summarized as follows : 1. The thermal changes of the area which is a meridian point in the Large Intestine Meridian of the acupuncture stimulation group on Hapkok different from the control groups with significantly change. 2.The thermal changes of the area which is a meridian point in the Large Intestine Meridian of acupuncture stimulation groups on non-meridian point was not significantly different from the control group. 3. The thermal changes of the area which is a meridian point in the Large Intestine Meridian of the acupuncture stimulation group with Bu-rotation on Hapkok different from the control group with significant increase. 4. The thermal changes of the area which is a meridian point in the Large Intestine Meridian of acupuncture stimulation group with Bu-rotation on non-meridian point was not significantly different from the control group. 5. The thermal changes of the area which is a meridian point in the Large Intestine Meridian of the acupuncture stimulation group with Xie-rotation on Hapkok different from the control group with significant decrease and increase following the decreasing or increasing temperature class, and the increasing temperature class of the acupuncture stimulation group with Xie-rotation on Hapkok significantly different from the acupuncture stimulation group with Bu-rotation on Hapkok. 6. The thermal changes of the area which is a meridian point in the Large Intestine Meridian of acupuncture stimulation group with Xie-rotation on non-meridian point was not significantly different from the control group. As a conclusion, I could think that the acupuncture stimulation with Bu-rotation or Xie-rotation on Hapkok affected the thermal change of the area which is a meridian point in the Large Intestine Meridian. And then I could relate these results with the existence of the meridian and meridian point, and with the Rotated Acupuncture-Bu-Xie theory of oriental medicine.

• Korean Journal of Acupuncture
• /
• v.33 no.2
• /
• pp.67-74
• /
• 2016

Objectives : The Knee degenerative osteoarthritis patients are not satisfied with the conventional therapies of KDOA, which results in the use of alternative therapies. The miniscalpel acupuncture is effective in treating chronic soft tissue, releasing contractures. However, there is little scientific evidence supporting the use of miniscalpel acupuncture in knee degenerative osteoarthritis. This study was designed to obtain basic data for a further large-scale trial as well as provide information about the feasibility of miniscalpel acupuncture in knee degenerative osteoarthritis patients. Methods : We describe the protocol for a randomized controlled pilot clinical trial of 5 weeks duration. Twenty patients will be recruited and randomly allocated to two treatment groups: miniscalpel acupuncture treatment(experimental group); and acupuncture and electro-acupuncture treatment(control group). Miniscalpel acupuncture will be performed once with a 1-week interval for 3 weeks. Electro-acupuncture will be administered twice per week for a period of 3 weeks. The primary outcomes will be measured by visual analogue scale and range of motion. The secondary outcomes will be short-form McGill Pain Questionnaire and Western Ontario and McMaster Universities Osteoarthritis Index. Both primary and secondary outcomes will be measured at baseline and at 1, 2, 3 and 5 weeks(i.e. 2 weeks after treatment completion). Conclusions : This pilot study will provide a basic foundation for a future large-scale trial as well as information about the feasibility of miniscalpel acupuncture in knee degenerative osteoarthritis.

• Journal of Life Science
• /
• v.25 no.4
• /
• pp.463-472
• /
• 2015

Adrenomedullin (AM) is a peptide expressed in all body tissues, and its related receptors are increased in liver fibrosis. In this study, we evaluated the effect of AM deficiency on liver fibrogenesis induced by $CCl_4$ using AM heterozygous (HT) mice. The animals received a single injection of $CCl_4$ or olive oil for the acute experiment, and received $CCl_4$ or olive oil three times a week for 6 weeks for the chronic experiment. Fibrosis was accessed using histopathological analysis and the western blot. The AM HT mice showed mild pericentrilobular degeneration when compared to the AM wild type (WT) mice. In the acute experiment, there was no significant difference between the AM WT and AM HT mice. However, in the chronic experiment, the $CCl_4$-treated AM HT mice showed more severe liver fibrosis than that of the CCl₄-treated AM WT mice. The AST and ALT levels of the AM HT $CCl_4$ group were higher than those of the AM WT CCl₄ group. Additionally, the collagen deposition, $\alpha$- SMA protein and TGF-$\beta$ protein were increased in the AM HT $CCl_4$ group when compared to the AM WT $CCl_4$ group. The AM HT mice also exhibited severe lipid peroxidation through the GSH decrement. Taken together, our data suggest that AM deficiency increases the susceptibility to liver fibrosis induced by $CCl_4$, indicating a novel therapeutic target for patients with liver fibrosis.

• Journal of Life Science
• /
• v.26 no.6
• /
• pp.663-672
• /
• 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
• /
• v.26 no.6
• /
• pp.633-639
• /
• 2016

Biomaterials including polymer, metal, ceramic, and composite have been widely applied for medical uses as medical fibers, artificial blood vessels, artificial joints, implants, soft tissue, and plastic surgery materials owing to their physicochemical properties. However, the biocompatibility and toxicity for film- and plate-form biomaterials is difficult to measure in mammalian cells because there is no appropriate incubation system. To solve these problems, we developed a novel mammalian cell culture system consisting of a silicone ring, top panel, and bottom panel and we applied two polymer films (PF) and one metal plate (MP). This system was based on the principal of sandwiching a test sample between the top panel and the bottom panel. Following the assembly of the culture system, SK-MEL-2 cells were seeded onto Styela Clava Tunic (SCT)-PF, NaHCO₃-added SCT (SCTN)-PF, and magnesium MP (MMP) and incubated at 37℃ for 24 hr and 48 hr. An MTT assay revealed that cell viability was maintained at a normal level in the SCT-PF culture group at 24 or 48 hr, although it rapidly decreased in the SCTN-PF culture group at 48 hr. Furthermore, the cell viability in the MMP culture group was very similar to that of the control group after incubation for 24 hr and 48 hr. Together, these results suggest the sandwich-type mammalian culture system developed here has the potential for the evaluation of the biocompatibility and toxicity of cells against PF- and MP-form biomaterials.

• Journal of Life Science
• /
• v.25 no.4
• /
• pp.433-440
• /
• 2015

Several studies suggest that regular consumption of walnuts may have beneficial effects against oxidative stress-mediated disease such as cancer. The present study reports the total phenolic and flavonoid contents, together with the antioxidant and antibacterial activities of several solvent extracts (methanol, n-hexane, ethyl acetate, n-butanol, and water) obtained from walnut (Juglans regia L.) green husk. MIC (minimal inhibitory concentration) values of the walnut extracts for 8 human pathogenic bacteria strain were determined using agar dilution method. Antioxidant activity of extracts were assessed using DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)) assays, EC₅₀ of DPPH and ABTS scavenging activities, and determination of total phenolic and flavonoid content and its correlation with DPPH and ABTS scavenging capacities. Among the six extracts, ethyl acetate extract (EtOAc Ex) showed the highest antimicrobial activity at 3.2 mg/ml of MICs against Staphylococcus aureus SG511. Total flavonoids and polyphenol contents of EtOAc Ex were 42.48 mg of quercetin equivalents (QE)/g and 223.25 mg of gallic acid equivalents (GAE)/g respectively. The highest antioxidative potential was shown by the sample extracted with EtOAc Ex (EC₅₀=13.43 μg/ml for DPPH and EC₅₀=41.83 μg/ml for ABTS radical scavenging activity assay). These results showed that J. regia green husk extracts can be used as an easily accessible source of natural antibacterial agents and natural antioxidants.

• Journal of Life Science
• /
• v.25 no.4
• /
• pp.456-462
• /
• 2015

We investigated the effects of fermented Sargassum thunbergii (FST) on platelet aggregation and serum lipid levels in rats made obese by a high fat diet. Six-week-old male SD-rats were randomly assigned to four groups as CON, HF-CON induced by high fat diet (HF), ST supplemented with HF (HFST100), and the fermented ST supplemented with HF group (HF-FST100). After 6 weeks, the results showed that the final weight and weight gain had decreased in the HF- FST100 group compared to the HF-CON group. Also, the food efficiency ratio was significantly reduced in the HF-FST100 group compared to HF-CON. The organ weights other than heart and spleen were significantly lower in the HF-FST100 group than in the HF-CON group. The levels of serum GOT and GPT significantly decreased in the HF-FST100 group over the HF-CON group. In addition, the total cholesterol, triglyceride and LDL-cholesterol levels were lower in the HF-FST100 group than in HF-CON, while the HDL-cholesterol level was higher in the HF-FST100. The ability of platelet aggregation of groups supplemented with FST was lower than the HF-CON group. These results suggest that FST may be beneficial in improving lipid profile and platelet aggregation in obesity.