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Purification of Human HtrA1 Expressed in E. coli and Characterization of Its Serine Protease Activity (E. coli에서 발현된 human HtrA1 단백질의 정제와 HtrA1의 serine protease 활성 조건에 관한 연구)

  • Kim, Kyung-Hee;Kim, Sang-Soo;Kim, Goo-Young;Rhim, Hyang-Shuk
    • 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.

Cytoprotective Effects of Schisandrin A against Hydrogen Peroxide-induced Oxidative Stress in SW1353 Human Chondrocytes (SW1353 인간 연골세포에서 산화적 스트레스에 대한 schisandrin A의 세포 보호 효과)

  • Jeong, Jin-Woo;Choi, Eun Ok;Kwon, Da Hye;Kim, Bum Hoi;Park, Dong Il;Hwang, Hye Jin;Kim, Byung Woo;Choi, Yung Hyun
    • 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.

A Reappraisal of Rural Public Service Location: the Case of Postal Facilities (農村地域의 郵政施設 立地問題)

  • Huh, Woo-Kung
    • Journal of the Korean Geographical Society
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    • v.31 no.1
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    • pp.1-18
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    • 1996
  • This study examines the spatial characteristics of postal office patronage in rural areas. in the light of future possible relocation and closures of the postal facilities. Most of private services have flown out small rural central places due to the decrease of supporting population, and there consequently remain only a few public services including government-run post offices at the Myon seats, the lowest level among rural central places in Korea. The small local population and its further decline undermine the rationale for maintaining such public services in depleted rural areas. For the worse of it, the government recently plans to transform the postal system to a quasi-private, corporational structure. One can fear that the profit-seeking nature of the new postal corporation will inevitably force to close many of such small rural facilities. The study first analysed nation-wide censuses of postal offices for the years of 1986 and 1992. The postal services examined are per capita number of postal stamps and revenue stamps sold, and letters, parcels, telegrams and monetary transactions handled at the post offices. It is found that, while the usage of postal services has increased substantially throughout the nation during the period of 1986-1992, the increment has largely been occurred by urban post offices rather than by those in Gun seats (i.e., rural counties); and that the gap of the service levels between urban and rural post offices is ever widening. The study further examined the service differentials among the post offices within rural counties to find that those post offices adjacent to the county (Gun) seats and larger urban centers rendered less amount of services than remote rural post offices, indicating that rural residents tend to partonize larger centers more and more than local Myon seats. At the second stage of the study, questionnaire surveys were conducted in Muju, Kimpo, and Hongsung-Gun's. These three counties are meant to represent respectively the remote, suburban, and intermediary counties in Korea. The analyses of survey data reveal that the postal hinterlands of the county seats extend to much of nearby Myons, the subdivisions of a Gun. It is also found that the extent of postal hinterlands of the three counties and the magnitude of patronage and quite different from each other depending upon the topography, population density, and the propinquity of the counties to metropolitan centers. The findings suggest to reappraise the current flat allocation scheme of public facilites to each of rural subdivisions throughout the nation. A detailed analysis on the travel behavior of the survey respondents yields that age is the most salient variable to distinguish activity spaces of rural residents. The activity spaces of older respondents tend to be more limited within their Myon, whereas those of younger respondents extend across the Myon boundary, toward the central towns and even distant larger cities. The very existence of several activity spaces in rural areas calls for an attention in the future locational decisions of public facilities. The locational criteria, employed by the Ministry of Communication of Korean government to establish a post office, are the size of hinterland population and the distance from adjacent postal facilities. The present study findings suggest two additional criteria: the order in rural central place hierarchy and the propinquity to the upper-level centers of the central hierarchy. These old and new criteria are complementary each other in that the former criteria are employed to determine new office locations, whereas the latter are appropriate to determine facility relocation and closures.

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Gene Expression of Surfactant Protein B and C in Endotoxin and Thiourea Treated Rats (내독소 및 Thiourea 투여 후 Surfactant protein B와 C 유전자 발현의 비교 관찰)

  • Sohn, Dong Hyun;Sohn, Jang Won;Yoon, Ho Joo;Shin, Dong Ho;Park, Sung Soo
    • Tuberculosis and Respiratory Diseases
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    • v.54 no.5
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    • pp.510-521
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    • 2003
  • Background : The surfactant specific proteins, SP-B and SP-C are believed to be important regulators of the surfactant function and homeostasis. Since acute respiratory distress syndrome(ARDS) is usually viewed as the functional and morphological expression of a similar underlying lung injury caused by a variety of insults, and since abnormalities in the surfactant function have been described in ARDS, the authors investigated the different effects of endotoxin and thiourea on the accumulation of mRNA encoding SP-B and SP-C. Methods : Sprague-Dawley rats were given 5 mg/kg of an intraperitoneal endotoxin from Salmonella enteritidis and 3.5 mg/kg intraperitoneal thiourea and were sacrificed at different time periods. Results : 1. The SP-B mRNA levels 6 and 24 hours after the 5 mg/kg endotoxin treatment was significantly reduced by 26.1% and 50%, respectively(P<0.01, P<0.001). 2. The SP-B mRNA levels 24 hours after the 3.5 mg/kg thiourea treatment was reduced by 9.8% and 12.5%, respectively. 3. The SP-C mRNA levels 6 and 24 hours after the 5 mg/kg endotoxin treatment was significantly reduced by 38.7% and 53.6%, respectively(P<0.01, P<0.001). 4. The SP-C mRNA level 6 hours after the 3.5 mg/kg thiourea treatment was reduced by 22.8%(P<0.05). Conclusion : These results indicate that the differential regulation of the hydrophobic surfactant proteins in vivo is evident, and suggest that the hydrophobic surfactant proteins might be differentially regulated during lung injury at different time periods without altering the lung wet to dry ratios. The mechanism of these alternations at the different time periods and the different kinds of etiology remain to be determined.

IS AN OXYGEN INHIBITION LAYER ESSENTIAL FOR THE INTERFACIAL BONDING BETWEEN RESIN COMPOSITE LAYERS? (Layering시 복합레진 층간의 계면 결합에서 oxygen inhibition layer가 필수적인가?)

  • Kim, Sun-Young;Cho, Byeong-Hoon;Baek, Seung-Ho;Lee, In-Bog
    • Restorative Dentistry and Endodontics
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    • v.33 no.4
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    • pp.405-412
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    • 2008
  • This study was aimed to investigate whether an oxygen inhibition layer (OIL) is essential for the interfacial bonding between resin composite layers or not. A composite (Z-250, 3M ESPE) was filled in two layers using two aluminum plate molds with a hole of 3.7 mm diameter. The surface of first layer of cured composite was prepared by one of five methods as followings, thereafter second layer of composite was filled and cured: Group 1 - OIL is allowed to remain on the surface of cured composite; Group 2 - OIL was removed by rubbing with acetone-soaked cotton; Group 3 - formation of the OIL was inhibited using a Mylar strip; Group 4 - OIL was covered with glycerin and light-cured; Group 5 (control) - composite was bulk-filled in a layer. The interfacial shear bond strength between two layers was tested and the fracture modes were observed. To investigate the propagation of polymerization reaction from active area having a photo-initiator to inactive area without the initiator, a flowable composite (Aelite Flow) or an adhesive resin (Adhesive of ScotchBond Multipurpose) was placed over an experimental composite (Exp_Com) which does not include a photoinitiator and light-cured. After sectioning the specimen, the cured thickness of the Exp_Com was measured. The bond strength of group 2, 3 and 4 did not show statistically significant difference with group 1. Groups 3 and 4 were not statistically significant different with control group 5. The cured thicknesses of Exp_Com under the flowable resin and adhesive resin were 20.95 (0.90) urn and 42.13 (2.09), respectively.

The Meaning Landscape of the Three Religion Consilience of Confucianism, Buddhism, and Taoism Embraced in Cheongamsa Temple, Gimcheon (김천 청암사에 수용된 유·불·선 삼교 통섭(統攝)의 의미경관)

  • Rho, Jae-Hyun;Kim, Hwa-Ok;Park, Yool-Jin;Kim, Young-Suk
    • Journal of the Korean Institute of Traditional Landscape Architecture
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    • v.35 no.1
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    • pp.57-67
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    • 2017
  • Based on the study on place name, meaning analysis of buddhist temple Palgyeng, and classifications of instructions and characteristics of writings on rocks, the main results of the study showed the following. Cheongamsa is located in the upstream of Muheulgugok Valley which was run by Hangang Jeong Gu(寒岡 鄭逑), a typical young man in the middle of the Joseon Dynasty, and overlaps with chief monk Byukam Lee Dukjin's(1896~?) object of Cheongamsa Palgyeng during Japanese colonial era. Including the mountain embracing Cheongamsa called Bullyeong-san, various characteristics of writings on rocks such as use of combination of place names including Sudosan, Seonlyeongsan, and Sinseondae, as well as Cheongamsa Temple, Bullyeongdongchun(佛靈洞天), Namuabitabul, Hogye(虎溪), Yeosan Waterfall(廬山瀑布), and Sejinam(洗 塵巖) show co-existence of Confucianism and Taoism mixed together in the temple. Especially for Cheongamsa Valley Hogye and Samsocheon(三笑泉) which is in the precincts of the temple, are realized as the symbol of the scenery of the three religion consilience of Confucianism, Buddhism, and Taoism through announcement using Hogyesamso(虎溪三笑) which originated from Yeosan Donglimsa, Jiangxi, China. Also, there are Yeosan Waterfall with a noble sense of place by negotiating with god, writing on rocks imprinted, and Yeosangyo(廬山橋) in Cheongamsa. As such, cultures of the three religion remain in various layers with the spirit of Hogyosamso symbolizing the consilience and coexistence of the three religion in Cheongamsa without the exclusiveness of Buddhism. Besides, the third precept of Yukhwagyeongbub, known to be practiced in Buddhist temples, which says "Do not only express self-assertion and ignore others" according to the dogma of working together in harmony, is in accordance with the spirits of Hogyesamso. As shown, Gimcheon Cheongamsa which is adorned with cultural landscapes of Hogyesamso, Gugokdongcheon(九曲洞天), and Palgyeng(八景), is not only good enough for the way of Buddhism and Confucianism but also for a place for the three religion consilience embracing the three religion.

Characteristics of Shear Strength and Elastic Waves in Artificially Frozen Specimens using Triaxial Compression Tests (삼축압축실험을 이용한 인공동결시료의 강도평가 및 탄성파 특성변화)

  • Kim, JongChan;Lee, Jong-Sub;Hong, Seung-Seo;Lee, Changho
    • The Journal of Engineering Geology
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    • v.24 no.1
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    • pp.111-122
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    • 2014
  • For accurate laboratory evaluations of soil deposits, it is essential that the samples are undisturbed. An artificial ground-freezing system is the one of the most effective methods for obtaining undisturbed samples from sand deposits. The objective of this study is to estimate the shear strengths and the characteristics of elastic waves of frozen-thawed and unfrozen specimens through the undrained triaxial compression test. For the experiments, Jumunjin standard sands are used to prepare frozen and unfrozen specimens with similar relative densities (60% and 80%). The water pluviation method is used to simulate the fully saturated condition under the groundwater table. When thawing the frozen specimens, the temperature is measured every minute. After the specimens are completely thawed, undrained triaxial compression tests are conducted using the same procedures as for the unfrozen specimens. During the triaxial tests (saturation, consolidation, and shear phase), compressional and shear waves are measured. The results show that the freeze-thaw process has minor effects on the peak deviatoric stress and shear strength values, and that the process does not affect the internal friction angle. The compressional wave velocity increases with increasing B-value to 1800 m/s in the saturation phase, but tends to remain constant in the process of consolidation and shearing. The shear wave velocity decreases with increasing B-value in the process of saturation, but changes velocity in accordance with the change in effective stress in the processes of consolidation and shearing. The compressional wave velocity has similar values regardless of the freeze-thaw process, but values of shear wave velocity are slighly lower in frozen-thawed specimens than in unfrozen specimens. This study is a preliminary experiment for estimating the shear strength and characteristics of elastic wave velocity in undisturbed frozen specimens that have been obtained using the artificial ground-freezing method.

Clinical Outcomes After Arthroscopic Double-Row Rotator Cuff Repair and Evaluation of Cuff Integrity by CT Arthrography (관절경적 2열 고정 회전근개 복원술 후의 임상 결과 및 CT 관절조영술을 이용한 건의 치유 평가)

  • Jo, Chris H.;Kim, Je-Kyoon;Yoon, Kang-Sup;Lee, Ji-Ho;Kang, Seung-Baek;Lee, Jae-Hyup;Han, Hyuk-Soo;Rhee, Seung-Whan
    • Clinics in Shoulder and Elbow
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    • v.12 no.2
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    • pp.199-206
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    • 2009
  • Purpose: Our goal for this study was to prospectively evaluate the functional & structural outcomes, by means of CT arthroscopy, of arthroscopic double-row fixation for treating rotator cuff tear. We also attempted to determine the variants that affect the functional & structural outcomes. Materials and Methods: Twenty seven consecutive patients underwent arthroscopic rotator cuff repair with double-row fixation. The average age at the time of the operation was fifty six years. The preoperative and postoperative examinations consisted of determining the Constant score, the score for the visual analogue scale for pain, the UCLA score, the American Shoulder and Elbow Surgeons (ASES) score, as well as a full physical examination of the shoulder. Preoperative MR arthrography was used to evaluate the integrity and atrophy of the rotator cuff. We measured the intraoperative tear size in the sagittal and coronal planes. Postoperative CT arthrography was used at one year postoperatively to evaluate the integrity and atrophy of the repaired tendons and muscles. Results: Preoperative MR arthrography revealed an average 29.22 mm tear size in the sagittal plane and an average 22.72 mm tear size in the coronal plane. Twelve cases of supraspinatus muscle atrophy and two cases of infraspinatus atrophy were observed on the preoperative MR arthrography. The average clinical outcome scores all significantly improved at the time of follow-up. At a mean of one year postoperatively, CT arthrography revealed 48.1% of the shoulders had healed, 11.1% showed incomplete healing and 40.7% showed retear of the repaired tendon. Conclusion: Arthroscopic double-row repair can result in improved clinical outcomes and good patient satisfaction. However, the problems about how to enhance healing of the repaired tendon still remain.

Heavy Metals and Pesticides with Maturing Process of Rehmanniae Radix Preparata (숙지황 제조과정에서 중금속과 잔류농약)

  • Kim, Jung-Ho;Yang, Joon-Young;Moon, Yung-Soo
    • Environmental Analysis Health and Toxicology
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    • v.22 no.3
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    • pp.255-261
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    • 2007
  • To obtain the risk assessment of hazardous materials with maturing process of Rehmanniae Radix Preparata, the residual heavy metals and pesticides in samples are surveyed. Concentration of As, Cd and Pb during the maturing process of Rehmanniae Radix Preparata of Korea are $0.147{\pm}0.007\;mg/L,\;0.025{\pm}0.001\;mg/L\;and\;0.193{\pm}0.021\;mg/L$, respectively. Concentration of As, Cd and Pb during the maturing process of Rehmanniae Radix Preparat of China are $0.0145{\pm}0.014\;mg/L,\;0.023{\pm}0.002\;mg/L\;and\;0.299{\pm}0.013\;mg/L$, respectively. Concentration of As, Cd and Hg remain unchanged during the maturing process of Rehmanniae Radix Preparata. Concentration of As, Cd and Pb in Rehmanniae Radix Preparat on the Korea are 3.53%, 6.66% and 5.16% for Korea Food & Drug Administration (KFDA) advisory level, respectively. The residual pesticides, such as aldrin, $BHC-{\alpha},\;BHC-{\beta},\;BHC-{\gamma},\;BHC-{\delta}$, captan, chlorfenapyr, chlorpyrifos, DDD, DDE, DDT-o,p, DDT-p.p, dieldrin, endosulfan-sulfate, $endosulfan-{\alpha}$, $endosulfan-{\beta}$, fenitrothion, penthoate, permethrin, quintozene, tolyfluanid and vinclozolin, are not detected in Rehmanniae Radix Preparat on the Korea and China. The hazardous materials in Rehmanniae Radix Preparata on the Korea and China are verified the safety of the residual heavy metals and pesticides compare with Korea Food & Drug Administration (KFDA) advistory level.

The Effects of Carbonate Minerals in Gully-pot Sediment on the Leaching Behavior of Heavy Metals Under Acidified Environment (우수관퇴적물에 함유된 탄산염광물이 산성환경에서의 중금속 용출거동에 미치는 영향 평가)

  • 이평구;유연희
    • Economic and Environmental Geology
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    • v.35 no.3
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    • pp.257-271
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    • 2002
  • One of the main interests in relation to heavily contaminated gully-pot sediment in urban area is the short term mobility of heavy metals, which depends on the pH of acidic rainwater and on the buffering effects of carbonate minerals. The buffering effects of carbonates are determined by titration (acid addition). Leaching experiments are carried out in solutions with variable initial HN03 contents for 24h. The gully-pot sediment appears to be predominantly buffered by calcite and dolomite. In case of sediment samples, which highly contain carbonates, pH decreases more slowly with increasing acidity. On the other hand, for the sediment samples, which less contain carbonate minerals, pH rapidly drops until it reaches about 2 then it decreases slowly. The leaching reactions are delayed until more acid is added to compensate for the buffering effects of carbonates. The Zn, Cu, Pb and Mn concentrations of leachate rapidly increase with decreased pH, while Cd, Co, Ni, Cr and Fe dissolutions are very slow and limited. The solubility of heavy metals depends not only on thc pH values of leachatc but also on the speciation in which metals are associated with sediment particles. In slightly to moderately acid conditions, Zn, Cd, Co, Ni and Cu dissolutions become increasingly important. As deduced from leaching runs, the relative mobility of heavy metals at pH of 5 is found to be: Zn > Cd > Co > Ni > Cu » Pb > Cr, suggesting that moderately acid rainwater leach Zn, Cd, Co, Ni and Cu from thc contaminated gully-pot sediment, while Pb and Cr would remain fixed. The buffering effects of Ca- and Mg-carbonates play an important role in delaying as well as limiting the leaching reactions of heavy metals from highly contaminated gully-pot sediment. The extent of such a secondary environmental pollution will thus depends on how well the metals in sediment can be leached by somewhat acidic rain water. Changes in the physicochemical environments may result in the severe environmental pollution of heavy metals. These results are to be taken into account in the management of contaminated sediments during rainstorms.