• 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.

• The Korean Journal of Food And Nutrition
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• v.28 no.2
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• pp.228-240
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• 2015

This study was conducted to investigate the dietary and other factors affecting bone mineral density (BMD) in older Korean women. A total of 340 women aged 65 to 74 were recruited from the Kugoksoondam area (Kurye, Goksung, Soonchang and Damyang counties), known as the longevity-belt region in Jeonla province, Korea. They were categorized into two groups according to bone status by T-score : a nonosteoporotic group and an osteoporotic group. Demographic characteristics were collected, as well as information on physical measurements, blood tests for biochemical indicators, health status health-related life style, dietary behavior, favorite food groups, nutrient intake and mini nutrition assessment (MNA). The results are as follows: The mean age of 185 nonosteoporotic women was 69.6 years and that of 155 osteoporotic women was 70.9 years (p<0.001). The mean T-score of the nonosteoporotic group was $-1.5mg/cm^3$ and that of theosteoporotic group was $-3.2mg/cm^3$ (p<0.001). Height and body weight in the nonosteoporotic group were significantly higher than in the osteoporotic group (p<0.001, respectively). There was no significant difference in BMI, although the BMI in the nonosteoporotic group was slightly higher. Waist and hip circumferences in the nonosteoporotic group were significantly higher than in the osteoporotic group (p<0.01, respectively), and the mid upper arm and calf circumferences were also significantly higher than in the osteoporotic group (p<0.001, p<0.01, respectively). The 5 m walking ability was significantly superior compared to the osteoporotic group. Serum levels did not show any significant differences between the groups and were within normal range. The serum total protein, albumin and Insulin-like growth factor (IGFs) levels of the nonosteoporotic group were significantly higher than those of the osteoporotic group (p<0.05, p<0.05, p<0.001, respectively). IGF was 104.7 ng/mL for the nonosteoporotic group and 88.1 ng/mL for the osteoporotic group. Physical activity and appetite in the nonosteoporotic group were significantly higher (p<0.01, p<0.05, respectively). The favorite food groups of the nonosteoporotic group comprised more meats and fish than those of the osteoporotic group (p<0.05, respectively). Nutrient intake was not significantly different, with the exception of niacin intake (p<0.05), but the nutrient intake of the nonosteoporotic group was slightly higher than that of the osteoporotic group. The niacin intake of the nonosteoporotic group and the osteoporotic group were 11.4 mgNE and 10.0 mgNE, corresponding to 103.6% and 90.9% of the Korean EAR, respectively. The MNA score of the nonosteoporotic group was significantly more favorable than for the osteoporotic group. In conclusion, it is necessary to maintain adequate body weight and muscle mass. Habitual physical activity may have a beneficial effect on BMD for older women. Dietary factors, such as meat and fish, higher intake of niacin rich foods and nutrient status for older women also appear to have favorable effects on bone mineral density.

• Journal of Life Science
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• v.33 no.2
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• pp.129-137
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• 2023

Reactive oxygen species (ROS) play an essential role in a variety of cellular physiological phenomena. The present study assessed the signaling axis that mediates the lysophosphatidic acid (LPA)-induced migration of SKOV-3 cells. Insulin-like growth factor-1 (IGF-1) stimulated SKOV-3 cell migration in a time- and dose-dependent manner. Similarly, LPA stimulated SKOV-3 cell migration and the phosphorylation of Akt in a time- and dose-dependent manner. The pharmacological inhibition of LPA receptors (LPA₁/LPA₃) significantly suppressed LPA-induced SKOV-3 cell migration. However, IGF-1-induced SKOV-3 cell migration was not affected by the inhibition of LPA1 and LPA3. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) or Rho-associated kinase (ROCK) significantly suppressed LPA-induced migration, whereas the inhibition of MAPK kinase (MEK) had no effect. Inhibition of PI3K or ROCK completely suppressed LPA-induced ROS generation, and suppression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) or chelation of ROS by N-acetylcysteine (NAC) blocked LPA-induced SKOV-3 cell migration. LPA-induced ROS generation was suppressed by silencing Rictor or Akt1 but not Raptor or Akt2. Silencing Rictor or Akt1 significantly suppressed LPA-induced SKOV-3 cell migration, whereas silencing Raptor or Akt2 had no effect. Finally, the overexpression of the constitutively active form Akt1 (CA-Akt1) significantly enhanced the LPA-induced migration of SKOV-3 cells. Given these results, we suggest that LPA stimulates SKOV-3 cell migration by ROS generation, which is mediated by the mTORC2/Akt1/NOX signaling axis.