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Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity

  • Kim, Young June;Ahn, Kwang Sung;Kim, Minjeong;Kim, Min Ju;Ahn, Jin Seop;Ryu, Junghyun;Heo, Soon Young;Park, Sang-Min;Kang, Jee Hyun;Choi, You Jung;Shim, Hosup
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.3
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    • pp.439-445
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    • 2017
  • Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

토양 및 지하수 Investigation 과 Remediation에 대한 현장적용

  • Wallner, Heinz
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2000.11a
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    • pp.44-63
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    • 2000
  • Situated close to Heathrow Airport, and adjacent to the M4 and M25 Motorways, the site at Axis Park is considered a prime location for business in the UK. In consequnce two of the UK's major property development companies, MEPC and Redrew Homes sought the expertise of Intergeo to remediate the contaminated former industrial site prior to its development. Industrial use of the twenty-six hectare site, started in 1936, when Hawker Aircraft commence aircraft manufacture. In 1963 the Firestone Tyre and Rubber Company purchased part of the site. Ford commenced vehicle production at the site in the mid-1970's and production was continued by Iveco Ford from 1986 to the plant's decommissioning in 1997. Geologically the site is underlain by sand and gravel, deposited in prehistory by the River Thames, with London Clay at around 6m depth. The level of groundwater fluctuates seasonally at around 2.5m depth, moving slowly southwest towards local streams and watercourses. A phased investigation of the site was undertaken, which culminated in the extensive site investigation undertaken by Intergeo in 1998. In total 50 boreholes, 90 probeholes and 60 trial pits were used to investigate the site and around 4000 solid and 1300 liquid samples were tested in the laboratory for chemical substances. The investigations identified total petroleum hydrocarbons in the soil up to 25, 000mg/kg. Diesel oil, with some lubricating oil were the main components. Volatile organic compounds were identified in the groundwater in excess of 10mg/l. Specific substances included trichloromethane, trichloromethane and tetrachloroethene. Both the oil and volatile compounds were widely spread across the site, The specific substances identified could be traced back to industrial processes used at one or other dates in the sites history Slightly elevated levels of toxic metals and polycyclic aromatic hydrocarbons were also identified locally. Prior to remediation of the site and throughout its progress, extensive liaison with the regulatory authorities and the client's professional representatives was required. In addition to meetings, numerous technical documents detailing methods and health and safety issues were required in order to comply with UK environmental and safety legislation. After initially considering a range of options to undertake remediation, the following three main techniques were selected: ex-situ bioremediation of hydrocarbon contaminated soils, skimming of free floating hydrocarbon product from the water surface at wells and excavations and air stripping of volatile organic compounds from groundwater recovered from wells. The achievements were as follows: 1) 350, 000m3 of soil was excavated and 112, 000m3 of sand and gravel was processed to remove gravel and cobble sized particles; 2) 53, 000m3 of hydrocarbon contaminated soil was bioremediated in windrows ; 3) 7000m3 of groundwater was processed by skimming to remove free floating Product; 4) 196, 000m3 of groundwater was Processed by air stripping to remove volatile organic compounds. Only 1000m3 of soil left the site for disposal in licensed waste facilities Given the costs of disposal in the UK, the selected methods represented a considerable cost saving to the Clients. All other soil was engineered back into the ground to a precise geotechnical specification. The following objective levels were achieved across the site 1) By a Risk Based Corrective Action (RBCA) methodology it was demonstrated that soil with less that 1000mg/kg total petroleum hydrocarbons did not pose a hazard to health or water resources and therefore, could remain insitu; 2) Soils destined for the residential areas of the site were remediated to 250mg/kg total petroleum hydrocarbons; in the industrial areas 500mg/kg was proven acceptable. 3) Hydrocarbons in groundwater were remediated to below the Dutch Intervegtion Level of 0.6mg/1; 4) Volatile organic compounds/BTEX group substances were reduced to below the Dutch Intervention Levels; 5) Polycyclic aromatic hydrocarbons and metals were below Inter-departmental Committee for the Redevelopment of Contaminated Land guideline levels for intended enduse. In order to verify the qualify of the work 1500 chemical test results were submitted for the purpose of validation. Quality assurance checks were undertaken by independent consultants and at an independent laboratory selected by Intergeo. Long term monitoring of water quality was undertaken for a period of one year after remediation work had been completed. Both the regulatory authorities and Clients representatives endorsed the quality of remediation now completed at the site. Subsequent to completion of the remediation work Redrew Homes constructed a prestige housing development. The properties at "Belvedere Place" retailed at premium prices. On the MEPC site the Post Office, amongst others, has located a major sorting office for the London area. Exceptionally high standards of remediation, control and documentation were a requirement for the work undertaken here.aken here.

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Transport Paths of Surface Sediment on the Tidal Flat of Garolim Bay, West Coast of Korea (황해 가로림만 조간대 표층퇴적물의 이동경로)

  • Shin, Dong-Hyeok;Yi, Hi-Il;Han, Sang-Joon;Oh, Jae-Kyung;Kwon, Su-Jae
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.3 no.2
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    • pp.59-70
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    • 1998
  • Two-dimensional trend-vector model of sediment transport is first tested in the tidal flat of Garolim Bay, mid-western coast of the Korean Peninsula. Three major parameters of surface sediment, i.e., mean grain size, sorting and skewness, are used for defining the best-fitting transport trend-vector on the sand ridge and muddy sand flat. These trend vectors are compared with the real transport directions determined from morphology, field observation and bedforms. The 15 possible cases of trend vectors are calculated from total sediments. In order to find the role of coarse sediments, trend vectors from sediments coarser than < 4.5 ${\phi}$, (sand size) are separately calculated from those of total sediments. As compared with the real directions, the best-fitting transport-vector model is the "case M" of coarse sediments which is the combined trend vectors of two cases: (1) finer, better sorted and more negatively skewed and (2) coarser, better sorted and more positively skewed. This indicates sand-size grains are formed by simpler hydrodynamic processes than total sediments. Transported sediment grains are better sorted than the source sediment grains. This indicates that consistent hydrodynamic energy can make sediment grains better sorted, regardless of complicated mechanisms of sediment transport. Consequently, both transported vector model and real transported direction show that the source of sediments are located outside of bay (offshore Yellow Sea) and in the baymouth. These source sediments are transported through the East Main Tidal Channel adjacent the baymouth. Some are transported from the subtidal zone to the upper tidal flat, but others are transported farther to the south, reaching the south tidal channel in the study area. Also, coarse sediment grains on the sand ridge are originally from the baymouth, and transported through the subtidal zone to the south tidal channel. These coarse sediments are moved to the northeast, but could not pass the small north tidal channel. It is interpreted that the great amount of coarse sediments is returned back to the outside of the bay (Yellow Sea) again through the baymouth during the ebb tide. The distribution of muddy sand in the northeastern part of study area may result from the mixing of two sediment transport mechanisms, i.e., suspension and bedload processes. The landward movement of sand ridge and the formation of the north tidal channel are formed either by the supply of coarse sediments originating from the baymouth and outside of the bay (subaqueous sand ridges including Jang-An-Tae) or by the recent relative sea-level rise.

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Tidal-Flat Sedimentation in a Semienclosed Bay with Erosional Shorelines: Hampyong Bay, West Coast of Korea (해안침식이 우세한 반폐쇄적 조간대의 퇴적작용: 한국 서해안의 함평만)

  • Chang, Jin-Ho;Kim, Yeo-Sang;Cho, Yeong-Gil
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.4 no.2
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    • pp.117-126
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    • 1999
  • Hampyong Bay is a semienclosed and macrotidal bay which opens to the eastern Yellow Sea through a narrow inlet in the southwestern coast of Korea. In order to understand the tidal-flat sedimentation in the semienclosed setting, morphology, sediments, accumulation rate and sea cliff erosion were investigated in the tidal flat of Hampyong Bay. The tidal flat of Hampyong Bay lacks intertidal drainage systems, and generally shows the concave-upward profile whose relief is designated by marked morphological features such as high-tide beaches, intertidal sand shoals and tidal creeks. Surfacial sediments of the tidal flat mainly consist of mud, sandy mud, gravelly mud, gravelly sand and muddy gravel, thus showing the textural characteristics of multimodal grain-size distribution, poorly sorting and positive skewness. The sediments generally coarsen landward due to the increase in coarse fraction content. Sedimentary structures are deeply bioturbated, but parallel lamination and lenticular bedding are locally found in the mudflat near mean low water line. Annual accumulation rates across the tidal flat (along Line SM) average -5.2 cm/yr with a range of -45.8~+4.2 cm/yr, indicating that the tidal flat is erosional. In general, erosion rates of upper and lower tidal flat are higher than those of middle tidal flat. Seasonally, the erosion rates are much higher during spring and winter when dominant wind direction corresponds to the long axis of Hampyong Bay. Sea cliffs are eroded at a rate of 1.4 m/yr. The biggest sea cliff erosion generally occurs 1~2 months later after tidal flats were extensively eroded. Such erosions of tidal Oats and sea cliffs in the semienclosed bay setting are interpreted to be due to wind waves coupled with local sea-level rise.

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Care Needs of Hospice Patients in Comparison with Those from the Family Caregivers' Perspective: Q methodology (호스피스 환자의 돌봄 요구와 가족이 인지하는 환자의 돌봄 요구 비교: Q 방법론)

  • Yong, Jin-Sun;Hong, Hyun-Ja
    • Journal of Hospice and Palliative Care
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    • v.7 no.2
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    • pp.153-168
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    • 2004
  • Purpose: The purpose of tile study was to identify types of care needs of hospice patients and those from the family caregivers' perspective and to compare these two groups in reporting patients' care needs through Q-methodology. Methods: Twenty three Q-statements concerning care needs were selected through in-depth interviews of hospice patients. Data were collected from 20 hospice patients as well as 20 family caregivers respectively by sorting 23 Q-statements into 9 points standard. Data analysis was performed by using PC QUANL program. Results: Principal component analysis identified four types of care needs of the hospice patients. Overall, the accuracy of family caregiver reports was 48% in all types of care needs. Type 1 was named 'physical care needs type' for those whose greatest need was physical care to be free of pain and comfortable. The accuracy in Type 1 was 62.5%. Type 2 was named 'emotional care needs type' for those who would like to share love and intimacy with their family members. The accuracy in Type 2 was 20%. Type 3 was named 'spiritual care needs type' for those who would like to receive forgiveness from their God and prayers and visitation of clergy. The accuracy in Type 3 was 60%. Type 4 was named 'social care needs type' for those who would like to complete their ongoing work and to give service to others. The accuracy in Type 4 was 50%. Conclusion: There was a great difference between hospice patients and the family caregivers in reporting patients' care needs. Thus, hospice nurses need to educate family caregivers to more accurately assess patients' care needs.

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Market Segmentation of Converging New Media Advertising: The Interpretative Approach Based on Consumer Subjectivity (융합형 뉴미디어 광고의 시장세분화 연구: 소비자 주관성에 근거한 해석적 관점에서)

  • Seo, Kyoung-Jin;Hwang, Jin-Ha;Jeung, Jang-Hun;Kim, Ki-Youn
    • Journal of Internet Computing and Services
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    • v.15 no.4
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    • pp.91-102
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    • 2014
  • The purpose of this research is to perform the consumer typological study of integrated emerging digital advertisement, where IT and advertisement industry were fused, and to propose the theoretical definition about consumer characteristic which is in need for collection of related market subdivision strategy in perspective of business marketing. For this, the Q methodology, the 'subjectivity' research of qualitative perspective, which discovers new theory by interpreting subjective system of thinking, preference, opinion, and recognition of inner side of respondents, was applied and analyzed. Compared to previous quantitative research that pursues hypothesis verification, this Q methodology is not dependent on operational definition proposed by researcher but pursues for analytic study completely reflecting objective testimony of respondents. For this reason, Q study analyzes in-depth the actual consumer type, which can be found at the initial market formation stage of new service, therefore this study is applicable for theorizing the consumer character as a mean of advanced research. This study extracted thirty 'IT integrated digital advertisement type (Q sample)' from thorough literature research and interviews, and eventually discovered a total four consumer types from analyzing each Q sorting research data of 40 respondents (P sample). Moreover, by interpreting subdivided intrinsic characteristic of each group, the four types were named as 'multi-channel digital advertisement pursuit type', 'emotional advertisement pursuit type', 'new media advertisement pursuit type', and Web 2.0 advertisement pursuit type'. The analysis result of this study is being expected for its value of usage as advanced research of academic and industrial research with the emerging digital advertisement industry as a subject, and as basic research in the field of R&D, Marketing program and the field of designing the advertisement creative strategy and related policy.

Comparative Study on the Viability of Frozen-thawed Primordial Germ Cells using Vitrification in Chicken Breed (초자화 동결법을 이용한 닭 품종간의 원시생식세포 동결성적의 비교)

  • Kim, Hyun;Kim, Dong Hun;Han, Jae Yong;Choi, Sung Bok;Ko, Yeoung Gyu;Do, Yoon Jung;Seong, Hwan Hoo;Kim, Sung Woo
    • Korean Journal of Poultry Science
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    • v.40 no.3
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    • pp.207-216
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    • 2013
  • This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

Isolation and characterization of human dental tissue-derived stem cells in the impacted wisdom teeth: comparison of dental follicle, dental pulp, and root apical papilla-derived cells (미성숙 매복지치의 치낭, 치수, 치근유두 조직에서 다능성 줄기세포의 분리와 특성화에 대한 연구)

  • Song, Jung-Ho;Park, Bong-Wook;Byun, June-Ho;Kang, Eun-Ju;Rho, Gyu-Jin;Shin, Sang-Hun;Kim, Uk-Kyu;Kim, Jong-Ryoul
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.36 no.3
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    • pp.186-196
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    • 2010
  • Introduction: The first aim of this study was to isolate the dental tissue-derived stem cells from the dental follicle (DF), dental pulp (DP), and root apical papilla (RAP) of the extracted wisdom teeth. Second was to evaluate their characterization with the expressions of transcription factors and cell surface markers. Finally, their ability of the in vitro multi-lineage differentiations into osteogenic and adipogenic cells were compared, respectively. Materials and Methods: Dental tissues, including dental follicle, dental pulp, and root apical papilla, were separated in the extracted wisdom teeth. These three dental tissues were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with supplements, respectively. After passage 3, the homogeneous shaped dental tissue-derived cells were analyzed the expression of transcription factors (Oct-4, Nanog and Sox-2) and cell surface markers (CD44, CD90 and CD105) with reverse transcription polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. In order to evaluate in vitro multi-lineage differentiations, the culture media were changed to the osteogenic and adipogenic induction mediums when the dental tissue-derived cells reached to passage 3. The characteristics of these three dental tissue-derived cells were compared with immunohistochemistry. Results: During primary culture, heterogenous and colony formatted dental tissue-derived cells were observed in the culture plates. After passage 2 or 3, homogenous spindle-like cells were observed in all culture plates. Transcription factors and mesenchymal stem cell markers were positively observed in all three types of dental tissue-derived cells. However, the quantity of expressed transcription factors was most large in RAP-derived cells. In all three types of dental tissue-derived cells, osteogenic and adipogenic differentiations were observed after treatment of specific induction media. In vitro adipogenic differentiation was similar among these three types of cells. In vitro osteogenic differentiation was most strongly and frequently observed in the RAP-derived cells, whereas rarely osteogenic differentiation was observed in the DP-derived cells. Conclusion: These findings suggest that three types of human dental tissue-derived cells from extracted wisdom teeth were multipotent mesenchymal stem cells, have the properties of multi-lineage differentiations. Especially, stem cells from root apical papilla (SCAP) have much advantage in osteogenic differentiation, whereas dental follicle cells (DFCs) have a characteristic of easy adipogenic differentiation.

Effect of Hypoxia-induced XIAP Expression on Apoptosis of Trophoblast Cells in Placenta (Hypoxia에 의한 X-linked Inhibitor of Apoptosis 발현이 태반 내 영양막세포의 세포자멸사에 미치는 영향)

  • Lee, Jong-Sung;Jeon, Su-Yeon;Choi, Jong-Ho;Lee, Yoo-Jin;Cha, Dong-Hyun;Kim, Gi-Jin
    • Clinical and Experimental Reproductive Medicine
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    • v.37 no.3
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    • pp.217-229
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    • 2010
  • Objective: Apoptosis plays an important role for the maintenance of the normal pregnancy. Expression of X-linked inhibitor of apoptosis (XIAP) is able to effectively prevent apoptosis and controls trophoblast cells death throughout placental development, but it is still unknown in the function of XIAP in trophoblast cells exposed to hypoxic condition, which is one of the factors causing preeclampsia. Therefore, we conducted to compare XIAP expression in normal and pre-eclamptic placenta tissues and analyzed the function of XIAP in HTR-8/SVneo trophoblast cell line exposed to hypoxic condition. Methods: The expression of XIAP was analyzed in placental tissues from the following groups of patients (none underwent labor): 1) term normal placenta (n=15); 2) term with pre-eclamptic placeneta (n=15); and 3) pre-term with pre-eclamptic placenta (n=11) using semi-quantitative RT-PCR, immunohistochemistry, and Western blot. In order to evaluate the function of XIAP in HTR-8/SVneo trophoblast cells under hypoxic condition, HIF-$1{\alpha}$ plasmids, and hypoxic condtion were transfected and treated into HTR-8/SVneo trophoblast cells for 24 hours, respectively. Results: We observed that XIAP are expressed in the syncytiotrophoblasts and syncytial knot of placental villi. The expression of XIAP was significantly decreased in preeclamptic placenta tissues than in normal placenta tissues without labor (p<0.05). Furthermore, we confirmed the XIAP expression in HTR-8/SVneo trophbolast cells exposed to hypoxia was translocated from cytoplasm into nucleus and decreased XIAP by hypoxic condition induced apoptosis in HTR-8/SVneo trophoblast cells through up-regulation of pro-apoptotic proteins. Conclusion: These results suggest that the expression of XIAP is involved in placental development as well as decreased expression of XIAP by hypoxia is associated with pre-eclampsia through inducing trophoblast cells apoptosis.

Identification of Quaternary Faults and shallow gas pockets through high-resolution reprocessing in the East Sea, Korea (탄성파 자료 고해상도 재처리를 통한 동해해역의 제4기 단층 및 천부 가스 인지)

  • Jeong, Mi Suk;Kim, Gi Yeong;Heo, Sik;Kim, Han Jun
    • Journal of the Korean Geophysical Society
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    • v.2 no.1
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    • pp.39-44
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    • 1999
  • High-resolution images are drawn from existing seismic data which were originally obtained by Korea Ocean Research & Development Institute (KORDI) during 1994-1997 for deep seismic studies on the East Sea of Korea. These images are analyzed for mapping Quaternary faults and near-bottom gas pockets. First 12 channels are selected from shot gathers for reprocessing. The processing sequence adopted for high-resolution seismic images comprises data copy, trace editing, true amplitude recovery, common-midpoint sorting, initial muting, prestack deconvolution, bandpass filtering, stacking, highpass filtering, poststack deconvolution, f-x migration, and automatic gain control (AGC). Among these processing steps, predictive deconvolution, highpass filtering, and short window AGC are the most significant in enhancement of resolution. More than 200 Quaternanry faults are interpreted on the migrated sections in the shallow depths beneath the seafloor. Although numerous faults are found mostly at the western continental slope and boundaries of the Ulleung Basin, significant amount of the faults are also indicated within the basin. Many of these faults are believed to be formed with reactivation of basement, from geotectonic activities including volcanism, and often originated in Tertiary, indicating that the tectonic regime of the East Sea might be unstable. Existence of shallow gas pockets casts real hazardous warnings to deep-sea drillings and/or to underwater constructions such as inter-island cables and gas pipelines. On the other hand, discovery of these gas pockets heightens the interests in developing natural resources in the East Sea. Reprocessed seismic sections, however, show no typical seismic characteristics for gas hydrates such as bottom-simulating reflectors in the western continental slope and ocean floor.

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