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Effects of Different Types of Dietary Fat on Muscle Atrophy According to Muscle Fiber Types and PPAR${\delta}$ Expression in Hindlimb-Immobilized Rats (지방의 종류가 다른 식이의 섭취가 하지고정 흰 쥐의 근 섬유별 근 위축과 PPAR${\delta}$ 활성에 미치는 영향)

  • Lee, Ho-Uk;Park, Mi-Na;Lee, Yeon-Sook
    • Journal of Nutrition and Health
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    • v.44 no.5
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    • pp.355-365
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    • 2011
  • This study investigated how dietary fat affects muscle atrophy and lipid metabolism in various muscles during hindlimb immobilization in rats. Twenty-four male Sprague?Dawley rats had their left hindlimb immobilized and were divided into four groups by dietary fat content and composition. The contralateral hindlimb (control) was compared with the immobilized limb in all dietary groups. Rats (n = 6/group) were fed a 4% corn oil diet (CO), 2.6% corn oil + 1.4% fish oil diet (FO), 30% corn oil diet (HCO), or a 30% beef tallow diet (HBT)after their hind limbs were immobilized for 10 days. Data were collected for the gastrocnemius, plantaris and soleus muscles. Muscle atrophy was induced significantly after 10 days of hindlimb immobilization, resulting in significantly decreased muscle mass and total muscle protein content. The protein levels of peroxisome proliferator activated receptor ${\delta}$ (PPAR${\delta}$) in the plantaris, gastrocnemius, and soleus increased following hindlimb immobilization irrespective of dietary fat intake. Interestingly, the PPAR${\delta}$ mRNA level in the plantaris decreased significantly in all groups and that in the FO group was lower than that in the other groups. The soleus PPAR${\delta}$ mRNA level decreased significantly following hindlimb immobilization in the FO group only. Muscle carnitine palmitoyl transferase 1 (mCPT1) mRNA level was not affected by hindlimb immobilization. However, the mCPT1 mRNA level in the FO group was significantly lower in the plantaris but higher in the soleus than that in the other groups. The pyruvate dehydrogenase kinase 4 (PDK4) mRNA level in the plantaris decreased significantly, whereas that in the soleus increased significantly following hindlimb immobilization. The plantaris, but not soleus, PDK4 mRNA level was significantly higher in the FO group than that in the CO group. The increased PPAR${\delta}$ protein level following hindlimb immobilization may have suppressed triglyceride accumulation in muscles and different types of dietary fat may have differentially affected muscle atrophy according to muscle type. Our results suggest that ${\omega}$-3 polyunsaturated fatty acids may suppress muscle atrophy and lipid accumulation by positively affecting the expression level and activity of PPAR${\delta}$ and PPAR${\delta}$-related enzymes, which are supposed to play an important role in muscle lipid metabolism.

The Determination of Trust in Franchisor-Franchisee Relationships in China (중국 프랜차이즈 시스템에서의 본부와 가맹점간 신뢰의 영향요인)

  • Shin, Geon-Cheol;Ma, Yaokun
    • Journal of Global Scholars of Marketing Science
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    • v.18 no.2
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    • pp.65-88
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    • 2008
  • Since the implementation of economic reforms in 1978, the Chinese economy grows rapidly at an average annul growth rate of 9% over the post two decades. Franchising has been widely recognized as an important source of entrepreneurial activity. Trust is important in that it facilitates relational exchanges by permits partners to transcend short-run inequities or risks to concentrate on long-term profits or gains. In the relationship between the franchisors and franchisees, trust has been described as an important source of competitive advantage. However, little research has been done on the factors affecting trust in Chinese franchisor-franchisee relationships. The purpose of this study is to investigate what factors affect the trust in the franchise system in China, and to provide guidelines and insights to franchisors which enter Chinese market. In this study, according to Morgan and Hunt (1994), trust is defined as the extending when one party has confidence in an exchange partner's reliability and integrity. We offered a conceptual model of the empirical study. The model shows that the factors affecting the trust include franchisor's supports, communication, satisfaction with previous outcome and conflict. We also suggested the franchisor's supports and communication like to enhance the franchisee's satisfaction with previous outcome, and the franchisor's supports, communication and he franchisee's satisfaction with previous outcome tend to decrease conflict. Before the formal study, a pretest involving exploratory interviews with owners from three franchisees was conducted to make sure the questionnaire was relevant and clear to the respondents. The data were collected using trained interviewers to carry out personal interviews with the aid of an unidentified, muti-page, structured questionnaire. The respondents comprised of owners, managers, and owner managers of franchisee-owned food service franchises located in Beijing, China. Even though a total of 256 potential franchises were initially contacted, the finally usable sample consisted of 125 respondents. As expected, the sampling method was successful in soliciting respondents with waried personal and firm characteristics. Self-administrated questionnaires were used for all measures. And established scales were used to measure the latent constructs in this study. The measures tapped the franchisees' perceptions of the relationship with the referent franchisor. Five-point Likert-type scales ranging from "strongly disagree" (=1) to "strongly agree" (=7) were used throughout the constructs (trust, eight items; support, five items; communication, four items; satisfaction, six items; conflict, three items). The reliability measurements traditionally employed, such as the Cronbach's alpha, were used. All the reliabilities were greater than.80. The proposed measurement model was estimated using SPSS 12.0 and AMOS 5.0 analysis package. We conducted A series of exploratory factor analyses and confirmatory factor analyses to assess the convergent validity, discriminant validity, and reliability. The results indicate reasonable overall fits between the model and the observed data. The overall fit of measurement model were $X^2$= 159.699, p=0.004, d.f. = 116, GFI =.879, NFI =.898, CFI =.969, IFI =.970, TLI =.959, RMR =.058. The results demonstrated that the data reasonably fitted the model. We also examined construct reliability and reliability and average variance extracted (AVE). The construct reliability of each construct was greater than.80 and the AVE of each construct was greater than.50. According to the analysis of Structure Equation Modeling (SEM), the results of path model indicated an adequate fit of the model: $X^2$= 142.126, p = 0.044, d.f. = 115, GFI =.892, NFI =.909, CFI =.981, IFI =.981, TLI =.974, RMR =.057. As hypothesized, the results showed that it is strategically important to establish trust in a franchise system, and the franchisor's supports, communication and satisfaction with previous outcome tend to reinforce franchisee's trust. The results also showed trust seems to decrease as the experience of conflict episodes increases. And we also noticed that franchisor's supports and communication tend to enhance the franchisee's satisfaction with previous outcome, and communication tend to decrease conflict. If the trust between the franchisor and franchisee can be established in a franchise system, franchising offers many benefits and reduces many costs. To manage a mutual trust of relationship with their franchisees, franchisor's should provide support effectively to their franchisees. Effective assistant services have direct effect on franchisees' satisfaction with previous outcome and trust in franchisor. Especially, franchise sales process, orientation, and training in the start-up period are key elements for success of the franchise system. Franchisor's support is an accumulated separate satisfaction evaluation with different kind of service provided by the franchisor. And providing support definitely can improve the trustworthy image of the franchisor. In the franchise system, conflicts of interests and exertions of different power sources are very common. The experience of conflict episodes seems to negatively relate to trust. Therefore, it is important to reduce the negative side of the relationship conflicts. Communication actually plays a broader role in reducing conflict and establish mutual trust in franchisor-franchisee relationship. And effective communication between franchisors and franchisees can improve franchisees' satisfaction toward the franchise system. As the diversification of Chinese markets, both franchisors and franchisees must keep the relevant, timely, and reliable communication. And it is very important to improve the quality of communication. Satisfaction with precious outcomes seems to positively relate to trust. Franchisors and franchisees that are highly satisfied with the previous outcomes that flow from their relationship will perceive their partner as advancing their goal achievement. Therefore, it is necessary for both franchisor and their franchisees to make the welfare of partner with effort. Little literature has focused on what factors affect the trust between franchisors and their franchisees in China. This study developed the hypotheses regarding the factors affecting trust in the transaction relationship. The results of data analysis supported the hypotheses strongly. There are certain limitations in this study. First, we may point out that some other factors missed in this study could be significantly important. Second, the context of this study, food service industry, limits its potential generalizability for all franchise systems. More studies in different categories of franchise system are needed to broaden its generalizability. Third, the model was tested empirically in a sample in Beijing, more empirical tests of the proposed model in other Chinese areas are needed. Finally, the analysis in this study was solely based on the perception of franchisees and the opinions of franchisors were not included.

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In vitro Antioxidant and Anti-Inflammatory Activities of Ethanol Extract and Sequential Fractions of Flowers of Prunus persica in LPS-Stimulated RAW 264.7 Macrophages (복숭아꽃 에탄올 추출물과 분획물의 in vitro 항산화 효과 및 RAW 264.7 대식세포에서의 항염증 효과)

  • Kwak, Chung Shil;Choi, Hye-In
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.44 no.10
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    • pp.1439-1449
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    • 2015
  • Prunus persica Flos (PPF) were investigated for their antioxidant and anti-inflammatory activities to find a natural functional food resource preventing degenerative diseases associated with excessive oxidative stress and chronic inflammation. PPF was extracted using ethanol (EtOH) and then sequentially fractioned by hexane (Hx), dichloromethane (DM), ethyl acetate (EA), n-butanol (BtOH), and water (DW). Contents of total phenolics and flavonoids, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2, and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-${\alpha}$ production were also measured using LPS-treated RAW 264.7 macrophages. EtOH extract showed relatively high antioxidant activity with high total phenolic (78.1 mg tannic acid/g) and flavonoid contents (55.3 mg rutin/g). EA fraction contained the highest total phenolic and flavonoid contents (394.6 mg tannic acid/g, 253.7 mg rutin/g), followed by BtOH (128.3 mg tannic acid/g, 93.1 mg rutin/g). EA and BtOH fractions and EtOH extract showed higher DPPH radical and ABTS radical scavenging activities than the others (P<0.05). In LPS-treated RAW 264.7 macrophages, EtOH extract ($200{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, and TNF-${\alpha}$ production levels to 38.5%, 32.3%, and 48.9% of the control, respectively, as well as reduced iNOS and COX-2 protein expression. DM fraction ($50{\mu}g/mL$) showed significantly reduced (P<0.05) NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 43.5%, 13.3%, 38.7%, and 41.3% of the control, respectively, and EA fraction ($50{\mu}g/mL$) showed significantly reduced NO, PGE2, IL-6, and TNF-${\alpha}$ production levels to 44.8%, 22.4%, 45.7%, and 62.0% of the control, respectively. Taken together, EtOH extract of PPF showed potent antioxidant and anti-inflammatory activities, and EA and BtOH fractions showed comparatively stronger antioxidant activities while DM and EA fractions showed stronger anti-inflammatory activities. It can be concluded that EtOH extract of PPF and its fractions are good candidates as natural resources for the development of anti-oxidative and anti-inflammatory functional food products.

Wind and Flooding Damages of Rice Plants in Korea (한국의 도작과 풍수해)

  • 강양순
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.34 no.s02
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    • pp.45-65
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    • 1989
  • The Korean peninsular having the complexity of the photography and variability of climate is located within passing area of a lots of typhoon occurring from the southern islands of Philippines. So, there are various patterns of wind and flooding damages in paddy field occuring by the strong wind and the heavy rain concentrated during the summer season of rice growing period in Korea. The wind damages to rice plants in Korea were mainly caused by saline wind, dry wind and strong wind when typhoon occurred. The saline wind damage having symptom of white head or dried leaves occurred by 1.1 to 17.2 mg of salt per dry weight stuck on the plant which was located at 2. 5km away from seashore of southern coastal area during the period(from 27th to 29th, August, 1986) of typhoon &Vera& accompanying 62-96% of relative humidity, more than 6 m per second of wind velocity and 22.5 to 26.4$^{\circ}C$ of air temperature without rain. Most of the typhoons accompanying 4.0 to 8. 5m per second of wind and low humidity (lesp an 60%) with high temperature in the east coastal area and southen area of Korea. were changed to dry and hot wind by the foehn phenomenon. The dry wind damages with the symptom of the white head or the discolored brownish grain occurred at the rice heading stage. The strong wind caused the severe damages such as the broken leaves, cut-leaves and dried leaves before heading stage, lodging and shattering of grain at ripening stage mechanically during typhoon. To reduce the wind damages to rice plant, cultivation of resistant varieties to wind damages such as Sangpoongbyeo and Cheongcheongbyeo and the escape of heading stage during period of typhoon by accelerating of heading within 15th, August are effective. Though the flood disasters to rice plant such as earring away of field, burying of field, submerging and lodging damage are getting low by the construction of dam for multiple purpose and river bank, they are occasionally occurred by the regional heavy rain and water filled out in bank around the river. Paddy field were submerged for 2 to 4 days when typhoon and heavy rain occurred about the end of August. At this time, the rice plants that was in younger growing stage in the late transplanting field of southern area of Korea had the severe damages. Although panicles of rice plant which was in the meiotic growing stage and heading stage were died when flooded, they had 66% of yield compensating ability by the upper tilling panicle produced from tiller with dead panicle in ordinary transplanting paddy field. It is effective for reduction of flooding damages to cultivate the resistant variety to flooding having the resistance to bacterial leaf blight, lodging and small brown planthopper simultaneously. Especially, Tongil type rice varieties are relatively resistant to flooding, compared to Japonica rice varieties. Tongil type rice varieties had high survivals, low elongation ability of leaf sheath and blade, high recovering ability by the high root activity and photosynthesis and high yield compensating ability by the upper tillering panicle when flooded. To minimize the flooding and wind damage to rice plants in future, following research have to be carried out; 1. Data analysis by telemetering and computerization of climate, actual conditions and growing diagnosis of crops damaged by disasters. 2. Development of tolerant varieties to poor natural conditions related to flooding and wind damages. 3. Improvement of the reasonable cropping system by introduction of other crops compensating the loss of the damaged rice. 4. Increament of utilization of rice plant which was damaged.

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Relationships between Micronutrient Contents in Soils and Crops of Plastic Film House (시설재배 토양과 작물 잎 중의 미량원소 함량 관계)

  • Chung, Jong-Bae;Kim, Bok-Jin;Ryu, Kwan-Sig;Lee, Seung-Ho;Shin, Hyun-Jin;Hwang, Tae-Kyung;Choi, Hee-Youl;Lee, Yong-Woo;Lee, Yoon-Jeong;Kim, Jong-Jib
    • Korean Journal of Environmental Agriculture
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    • v.25 no.3
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    • pp.217-227
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    • 2006
  • Micronutrient status in soils and crops of plastic film house and their relationship were investigated. Total 203 plastic film houses were selected (red pepper, 66; cucumber, 63; tomato, 74) in Yeongnam region and soil and leaf samples were collected. Hot-water extractable B and 0.1 N HCl extractable Cu, Zn, Fe, and Mn in soil samples and total micronutrients in leaf samples were analyzed. Contents Zn, Fe, and Mn in most of the investigated soils were higher than the upper limits of optimum level for general crop cultivation. Contents of Cu in most soils of cucumber and tomato cultivation were higher than the upper limit of optimum level, but Cu contents in about 30% of red pepper cultivation soils were below the sufficient level. Contents of B in most soils of cucumber and tomato were above the sufficient level but in 48% of red pepper cultivation soils B were found to be deficient. Micronutrient contents in leaf of investigated crops were much variable. Contents of B, Fe, and Mn were mostly within the sufficient levels, while in 71% of red pepper samples Cu was under deficient level and in 44% of cucumber samples Cu contents were higher than the upper limit of sufficient level. Contents of Zn in red pepper and cucumber samples were mostly within the sufficient level but in 62% of tomato samples Zn contents were under deficient condition. However, any visible deficiency or toxicity symptoms of micronutrients were not found in the crops. No consistent relationships were found between micronutrient contents in soil and leaf, and this indicates that growth and absorption activity of root and interactions among the nutrients in soil might be important factors in overall micronutrient uptake of crops. For best management of micronutrients in plastic film house, much attention should be focused on the management of soil and plant characteristics which control the micronutrient uptake of crops.

Pilot-scale Applications of a Well-type Reactive Barrier using Autotrophic Sulfur-oxidizers for Nitrate Removal (독립영양 황탈질 미생물을 이용한 관정형 반응벽체의 현장적용성 연구)

  • Lee, Byung-Sun;Um, Jae-Yeon;Lee, Kyu-Yeon;Moon, Hee-Sun;Kim, Yang-Bin;Woo, Nam-C.;Lee, Jong-Min;Nam, Kyoung-Phile
    • Journal of Soil and Groundwater Environment
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    • v.14 no.3
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    • pp.40-46
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    • 2009
  • The applicability of a well-type autotrophic sulfur-oxidizing reactive barrier (L $\times$ W $\times$ D = $3m\;{\times}\;4\;m\;{\times}\;2\;m$) as a long-term treatment option for nitrate removal in groundwater was evaluated. Pilot-scale (L $\times$ W $\times$ D = $8m\;{\times}\;4\;m\;{\times}\;2\;m$) flow-tank experiments were conducted to examine remedial efficacy of the well-type reactive barrier. A total of 80 kg sulfur granules as an electron donor and Thiobacillus denitrificans as an active bacterial species were prepared. Thiobacillus denitrificans was successfully colonized on the surface of the sulfur granules and the microflora transformed nitrate with removal efficiency of ~12% (0.07 mM) for 11 days, ~24% (1.3 mM) for 18 days, ~45% (2.4 mM) for 32 days, and ~52% (2.8 mM) for 60 days. Sulfur granules attached to Thiobacillus denitrificans were used to construct the well-type reactive barrier comprising three discrete barriers installed at 1-m interval downstream. Average initial nitrate concentrations were 181 mg/L for the first 28 days and 281 mg/L for the next 14 days. For the 181 mg/L (2.9 mM) plume, nitrate concentrations decreased by ~2% (0.06 mM), ~9% (0.27 mM), and ~15% (0.44 mM) after $1^{st}$, $2^{nd}$, and $3^{rd}$ barriers, respectively. For the 281 mg/L (4.5 mM) plume, nitrate concentrations decreased by ~1% (0.02 mM), ~6% (0.27 mM), and ~8% (0.37 mM) after $1^{st}$, $2^{nd}$, and $3^{rd}$ barriers, respectively. Nitrate plume was flowed through the flow-tank for 49 days by supplying $1.24\;m^3/d$ of nitrate solution. During nitrate treatment, flow velocity (0.44 m/d), pH (6.7 to 8.3), and DO (0.9~2.8 mg/L) showed little variations. Incomplete destruction of nitrate plume was attributed to the lack of retention time, rarely transverse dispersion, and inhibiting the activity of denitrification enzymes caused by relatively high DO concentrations. For field applications, it should be considered increments of retention time, modification of well placements, and intrinsic DO concentration.

Synthesis and Preliminary Evaluation of $9-(4-[^{18}F]Fluoro-3-hydroxymethylbutyl)$ Guanine $([^{18}F]FHBG)$ in HSV1-tk Gene Transduced Hepatoma Cell (9-(4-$[^{18}F]Fluoro-3-hydroxymethylbutyl)$guanine $([^{18}F]FHBG)$의 합성과 헤르페스 단순 바이러스 티미딘 키나아제 이입 간암 세포주에서의 기초 연구)

  • Moon, Byung-Seok;Lee, Tae-Sup;Lee, Myoung-Keun;Lee, Kyo-Chul;An, Gwang-Il;Chun, Kwon-Soo;Awh, Ok-Doo;Chi, Dae-Yoon;Choi, Chang-Woon;Lim, Sang-Moo;Cheon, Gi-Jeong
    • Nuclear Medicine and Molecular Imaging
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    • v.40 no.4
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    • pp.218-227
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    • 2006
  • Purpose: The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), $9-(4-[^{18}F]Fluoro-3-hydroxymethylbutyl)$guanine ($[^{18}F]FHBG$) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Materials and Methods: Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with $K[^{18}F]/K2.2.2.$ in acetonitrile using N2-monomethoxytrityl-9-14-(tosyl)-3-monomethoxytritylmethylbutyl]guanine as a precursor, followed by deprotection with 1 N HCl. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of $[^{18}F]FHBG$ were performed, and was analyzed correlation between $[^{18}F]FHBG$ uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor bearing Balb/c-nude mouse model. Results: $[^{18}F]FHBG$ was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiothemical yield was about 20-25%) (corrected for decay), radiochemical purity was >95% and specific activity was around >55.5 $GBq/{\mu}\;mol$. Specific accumulation of $[^{18}F]FHBG$ was observed in HSV1-tk gene transduced MCA-tk cells but not in MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked $[^{18}F]FHBG$ was retained inside of cells. The uptake of $[^{18}F]FHBG$ was showed a highly significant linear correlation ($R^2=0.995$) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. Conclusion: $[^{18}F]FHBG$ appears to be a useful as non-invasive PET imaging substrate in HSV1-tk expressing hepatoma model.

A Refined Method for Quantification of Myocardial Blood Flow using N-13 Ammonia and Dynamic PET (N-13 암모니아와 양전자방출단층촬영 동적영상을 이용하여 심근혈류량을 정량화하는 새로운 방법 개발에 관한 연구)

  • Kim, Joon-Young;Lee, Kyung-Han;Kim, Sang-Eun;Choe, Yearn-Seong;Ju, Hee-Kyung;Kim, Yong-Jin;Kim, Byung-Tae;Choi, Yong
    • The Korean Journal of Nuclear Medicine
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    • v.31 no.1
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    • pp.73-82
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    • 1997
  • Regional myocardial blood flow (rMBF) can be noninvasively quantified using N-13 ammonia and dynamic positron emission tomography (PET). The quantitative accuracy of the rMBF values, however, is affected by the distortion of myocardial PET images caused by finite PET image resolution and cardiac motion. Although different methods have been developed to correct the distortion typically classified as partial volume effect and spillover, the methods are too complex to employ in a routine clinical environment. We have developed a refined method incorporating a geometric model of the volume representation of a region-of-interest (ROI) into the two-compartment N-13 ammonia model. In the refined model, partial volume effect and spillover are conveniently corrected by an additional parameter in the mathematical model. To examine the accuracy of this approach, studies were performed in 9 coronary artery disease patients. Dynamic transaxial images (16 frames) were acquired with a GE $Advance^{TM}$ PET scanner simultaneous with intravenous injection of 20 mCi N-13 ammonia. rMBF was examined at rest and during pharmacologically (dipyridamole) induced coronary hyperemia. Three sectorial myocardium (septum, anterior wall and lateral wall) and blood pool time-activity curves were generated using dynamic images from manually drawn ROIs. The accuracy of rMBF values estimated by the refined method was examined by comparing to the values estimated using the conventional two-compartment model without partial volume effect correction rMBF values obtained by the refined method linearly correlated with rMBF values obtained by the conventional method (108 myocardial segments, correlation coefficient (r)=0.88). Additionally, underestimated rMBF values by the conventional method due to partial volume effect were corrected by theoretically predicted amount in the refined method (slope(m)=1.57). Spillover fraction estimated by the two methods agreed well (r=1.00, m=0.98). In conclusion, accurate rMBF values can be efficiently quantified by the refined method incorporating myocardium geometric information into the two-compartment model using N-13 ammonia and PET.

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Development of Dual Reporter System of Mutant Dopamine 2 Receptor ($D_2R$) and Sodium Iodide Symporter (NIS) Transgenes (변이 도파민 2 수용체와 나트륨 옥소 공동 수송체 이입유전자의 이중 리포터시스템 개발)

  • Hwang, Do-Won;Lee, Dong-Soo;Kang, Joo-Hyun;Chang, Young-Soo;Kim, Yun-Hui;Jeong, Jae-Min;Chung, June-Key;Lee, Myung-Chul
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.4
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    • pp.294-299
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    • 2004
  • Purpose: Both human NIS and mutant $D_2R$ transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor ($D_2R$) and compared its characteristics. Materials and Methods: The recombinant plasmid ($pIRES-hNIS/D_2R$) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter $pIRES-hNIS/D_2R$ was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND ($SK-Hep1-hNIS/D_2R$) cells stably expressing hNIS and $D_2R$ was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and $D_2R$ genes. The expressions of hNIS and $D_2R$ were measured by $^{125}I$ uptake assays and receptor binding assays. Specific binding of $D_2R$ to $[^3H]spiperone$ was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. $K_d\;and\;B_{max}$ values were estimated. The correlation between hNIS and $D_2R$ expression was compared by using each clone. Results: Similar quantities of hNIS and $D_2R$ genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. $^{125}I$ uptake in HEP-ND cells was completely inhibited by $KClO_4$, a NIS inhibitor Specific binding to HEP-ND cells was saturable and the $K_d\;and\;B_{max}$ values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and $D_2R$ binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and $D_2R$ in a stably transfected cell line. We expect that $D_2R$ and hNIS genes can complement mutually as a nuclear reporting system or that $D_2R$ can be used as reporter gene when hNIS gene were used as a treatment gene.

Radioiodine Therapy of Liver Cancer Cell Following Tissue Specific Sodium Iodide Symporter Gene Transfer and Assessment of Therapeutic Efficacy with Optical Imaging (조직 특이 발현 Sodium Iodide Symporter 유전자 이입에 의한 방사성옥소 간암세포 치료와 광학영상을 이용한 치료효과 평가)

  • Jang, Byoung-Kuk;Lee, You-La;Lee, Yong-Jin;Ahn, Sohn-Joo;Ryu, Min-Jung;Yoon, Sun-Mi;Lee, Sang-Woo;Yoo, Jeong-Soo;Cho, Je-Yeol;Lee, Jae-Tae;Ahn, Byeong-Cheol
    • Nuclear Medicine and Molecular Imaging
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    • v.42 no.5
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    • pp.383-393
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    • 2008
  • Purpose: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration of iodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. Materials and Methods: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with 1-131 was performed. In vivo nuclear imaging was obtained with gamma camera after 1-131 intraperitoneal injection. Results: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with 1-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. Conclusion: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of 1-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.