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Effect of $H_2O_2$ on Alveolar Epithelial Barrier Properties (폐상피세포 장벽에 대한 $H_2O_2$의 영향)

  • Suh, Duk-Joon;Cho, Se-Heon;Kang, Chang-Woon
    • Tuberculosis and Respiratory Diseases
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    • v.40 no.3
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    • pp.236-249
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    • 1993
  • Background: Among the injurious agents to which the lung airspaces are constantly exposed are reactive species of oxygen. It has been widely believed that reactive oxygen species may be implicated in the etiology of lung injuries. In order to elucidated how this oxidant causes lung cell injury, we investigated the effects of exogenous $H_2O_2$ on alveolar epithelial barrier characteristics. Methods: Rat type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with HEPES-buffered Ringer solution. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against $H_2O_2$ injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ, an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results: These monolayers have a high transepithelial resistance (>2000 ohm-$cm^2$) and actively transport $Na^+$ from apical fluid. $H_2O_2$(0-100 mM) was then delivered to either apical or basolateral fluid. Resulting indicated that $H_2O_2$ decreased Isc and R gradually in dose-dependent manner. The effective concentration of apical $H_2O_2$ at which Isc (or R) was decreased by 50% at one hour ($ED_{50}$) was about 4 mM. However, basolateral $H_2O_2$ exposure led to $ED_{50}$ for Isc (and R) of about 0.04 mM. Inhibition of cellular catalase yielded $ED_{50}$ for Isc (and R) of about 0.4 mM when $H_2O_2$ was given apically, while $ED_{50}$ for basolateral exposure to $H_2O_2$ did not change in the presence of ATAZ. The rate of $H_2O_2$ consumption in apical and basolateral bathing fluids was the same, while cellualr catalase activity rose gradually with time in culture. Conclusion: Our data suggest that basolateral $H_2O_2$ may affect directly membrane component (e.g., $Na^+,\;K^+$-ATPase) located on the basolateral cell surface. Apical $H_2O_2$, on the other hand, may be largely degraded by catalase as it passes through the cells before reaching these membrane components. We conclude that alveolar epithelial barrier integrity as measured by Isc and R are compromised by $H_2O_2$ being relatively sensitive to basolateral (and insensitive to apical) $H_2O_2$.

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The Effect of Surfactant on Neutrophil Apoptosis in Lipopolysaccharide Induced Acute Lung Injury in Rat (기관내 내독소 투여로 유도한 백서의 급성 폐손상 모델에서 surfactant가 호중구의 아포토시스에 미치는 영향)

  • Yoo, Ji-Hoon;Lee, Byoung-Jun;Jeong, Do-Young;Lee, Sang-Hoon;Shin, Jong-Wook;Kim, Jae-Yeol;Park, In-Won;Choi, Byoung-Whui
    • Tuberculosis and Respiratory Diseases
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    • v.53 no.4
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    • pp.409-419
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    • 2002
  • Background : The therapeutic effects of surfactant on acute lung injury derive not only from its recruiting action on collapsed alveoli but also from its anti-inflammatory effects. Pro-apoptotic action on alveolar neutrophils represents one of the important anti-inflammatory mechanisms of surfactant. In the present study, we evaluated the effects of sufactant on the apoptosis of human peripheral and rat alveolar neutrophils. Methods : In the (Ed- the article is not definitely needed but it helps to separate the two prepositions 'in') in vitro study, human neutrophils were collected from healthy volunteers. An equal number of neutrophils ($1{\times}10^6$) (Ed-confirm) was treated with LPS (10, 100, 1000ng/ml), surfactant (10, 100, $1000{\mu}g/ml$), or a combination of LPS (1000ng/ml) and surfactant (10, 100, $1000{\mu}g/ml$). After incubation for 24 hours, the apoptosis of neutrophils was evaluated by Annexin V method. In the in vivo study, induction of acute lung injury in SD rats by intra-tracheal instillation of LPS (5mg/kg) was followed by intra-tracheal administration of either surfactant (30mg/kg) or normal saline (5ml/kg). Tenty-four hours after LPS instillation, alveolar neutrophils were collected and the apoptotic rate was evaluated by Annexin V method. In addition, changes of the respiratory mechanics of rats (respiratory rate, tidal volume, and airway resistance) were evaluated with one chamber body plethysmography before, and 23 hours after, LPS instillation. Results : in the in vitro study, LPS treatment decreased the apoptosis of human peripheral blood neutrophils (control: $47.4{\pm}5.0%$, LPS 10ng/ml; $30.6{\pm}10.8%$, LPS 100ng/ml; $27.5{\pm}9.5%$, LPS 1000ng/ml; $24.4{\pm}7.7%$). The combination of low to moderate doses of surfactant with LPS promoted apoptosis (LPS 1000ng/ml + Surf $10{\mu}g/ml$; $36.6{\pm}11.3%$, LPS 1000ng/ml +Surf $100{\mu}g/ml$; $41.3{\pm}11.2%$). The high dose of surfactant ($1000{\mu}g/ml$) decreased apoptosis ($24.4{\pm}7.7%$) and augmented the anti-apoptotic effect of LPS (LPS 1000ng/ml + Surf $1000P{\mu}g/ml$; $19.8{\pm}5.4%$). In the in vivo study, the apoptotic rate of alveolar neutrophils of surfactant-treated rats was higher than that of normal saline-treated rats ($6.03{\pm}3.36%$ vs. $2.95{\pm}0.58%$). The airway resistance (represented by Penh) of surfactant-treated rats was lower than that of normal saline-treated rats at 23 hours after LPS injury ($2.64{\pm}0.69$ vs. $4.51{\pm}2.24$, p<0.05). Conclusion : Surfactant promotes the apoptosis of human peripheral blood and rat alveolar neutrophils. Pro-apoptotic action on neutrophils represents one of the important anti-inflammatory mechanisms of surfactant.

Study of the Impact of Light Through the Vitamin $B_{12}$/Folate Inspection (Vitamin $B_{12}$/Folate 검사 시 빛의 영향에 대한 고찰)

  • Cho, Eun Bit;Pack, Song Ran;Kim, Whe Jung;Kim, Seong Ho;Yoo, Seon Hee
    • The Korean Journal of Nuclear Medicine Technology
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    • v.16 no.2
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    • pp.162-166
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    • 2012
  • Purpose : Vitamin $B_{12}$ and Folate are for anemia work-up which is well known for its sensitivity of light; the screening manual also specifies to be careful with light conditions. According to this, our laboratory minimized the exposure of light when inspecting the Vitamin $B_{12}$ and Folate, but the exposure cannot be wholly blocked due to other various factors such as when conducting specimen segregation. Thus, this inspection is to identify to what extent light can influence and whether the exclusion of light is mandatory during the Vitamin $B_{12}$/Folate test. Materials and Methods : We have conducted two experiments of identifying the extent of light's influence when conducting the Vitamin $B_{12}$/Folate test and also when specimens are under preservation. These experiments were progressed with various concentrations of patients' specimens which were requested to our hospital in March 2012. The first experiment is to verify the results on Vitamin $B_{12}$/Folate dependent on light exposure during the experiment. In the process, we have compared the results of light exposure/exclusion during the incubation process after the reagent division. The second experiment is about the impact of light exposure on the results on Vitamin $B_{12}$/Folate during the preservation. For 1, 2, 7 days the light on the specimen were wholly blocked and were preserved under $-15^{\circ}C$ temperature refrigeration. Then, we compared the results of light-excluded specimen and the exposed one. Results : When conducting first experiment, there were no noticeable changes in the Standard and specimen's cpm, but for Vitamin $B_{12}$, the average result of specimen exposed to light increased 7.8% compare to that of excluded one's. Furthermore, in the significant level 0.05, the significance probability or the p-value was 0.251 which means it has no impact. For Folate, the result being exposed to light decreased 5.4%, the significance probability was 0.033 which means it has little impact. For the second preservation, the result was dependent on the light exposure. The first day of preservation of Vitamin $B_{12}$, the clinical material exposed to light was 11.6%, second day clinical material exposed to light was 10.8%, seventh day clinical material exposed to light increased 3.8%, the significance probability of the $1^{st}$, $2^{nd}$, $7^{th}$ day is 0.372, 0.033, 0.144 respectively, and which indicates that the $1^{st}$ and $7^{th}$ day seems to have no impact. For Folate's case, the clinical material exposed to light has increased 1.4% but hardly had impact, $2^{nd}$ day clinical material being exposed to light was 6.1%, $7^{Th}$ day clinical material being exposed to light decreased 5.2%. The significance probability of Folate on the $1^{st}$, $2^{nd}$, $7^{th}$ day is 0.378, 0.037, 0.217 respectively, and the $1^{st}$ day and the $7^{th}$ day seems to have no impact. Conclusion : After scrutinizing the impact of light exposure/exclusion, Vitamin $B_{12}$ has no impact, while Folate seems to have no noticeable influence but light exclusion is recommended due to its significance probability of 0.033 when conducting experiment. During the preservation, the $2^{nd}$ day result depend on the light exclusion seems to have impact or influence. However, to consider the complication of the experimental process, the experiment including technical errors is predictable. Hence, it is likely to have no impact of light. Nevertheless, it is recommendable to exclude the light during the long preservation as per the significance probability (p-value) of $1^{st}$ and $7^{th}$ day has been diminished.

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Evaluation of the Parameters of Soil Potassium Supplying Power for Predicting Yield Response, K2O Uptake and Optiumum K2O Application Levels in Paddy Soils (수도(水稻)의 가리시비반응(加里施肥反応)과 시비량추정(施肥量推定)을 위한 가리공급력(加里供給力) 측정방법(測定方法) 평가(評価) -I. Q/I 관계(関係)에 의(依)한 가리(加里) 공급력측정(供給力測定)과 시비반응(施肥反応))

  • Park, Yang-Ho;An, Soo-Bong;Park, Chon-Suh
    • Korean Journal of Soil Science and Fertilizer
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    • v.16 no.1
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    • pp.42-49
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    • 1983
  • In order to find out the possibility of predicting fertilizer K requirement from the K supplying capacity of soil, the relative K activity ratio, Kas/kai, the potential buffering capacity of $K^+$ ($PBC^k$ ; the liner regression coefficient) and its activity ratio ($AR^k_o$ ; $^{k+}$/${\sqrt{Ca^{+2}+Mg^{+2}}}$ in mol/l) at ${\delta}K$ = O, in the Q/I relationships of Beckett(1964), were determined for the soils before flooding and the samples taken at heading stage of transplanted rice in pot experiment. These parameters assumed as the K supplying capacity of soils were subjected for the investigation through correlation stady between themselves and other factors such as grain yield or the amounts of $K_2O$ uptake by rice plant at harvest. The results may be summarized as follows; 1. The potassium supplying power of the flooded soil was considered to be ruled by the amounts of exchangeable K before flooding, since there was little change in exchangeable K concentration from no-exchangeable K during the incubation periods of 67 days. 2. The $PBC^k$ values, in soils before flooding were 0.027, 0.014 and 0.009, where as the $AR^k_o{\times}10^{-3}$ values were 9.1, 7.6, and 15.4, respectively, in clay, loamy and sandy loam soils. 3. The $PBC^k$ values, determined in the soil samples taken at heading stage, varied little compared with the values of orignal soil, regardless of those different fertilizer treatments and textures, showing the possibility of using them as a factor for the improvement of soil to increase the efficiency of fertilizer K. 4. The significant yield responses to potassium fertilizer application were observed wherever the $AR^k_o$ values in soil at heading stage drop down to the original $AR^k_o$ values, regardless of any levels of fertilizer application. 5. The higher correlations between the gain yield or the amounts of $K_2O$ uptake and by the use of both soil factors of $PBC^k$ and $AR^k_o$ at heading stage were observed compared with the use of any single factor. 6. The Kas/Kai value in the soil, estimated prior to the experiment, had high possitive correlation with the $AR^k_o$ determined in the soil at heading stage and could be used as a soil factor for predicting potassium fertilizer requirement.

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Studies on Biology and Control of the Mulberry Small Weevil, Baris deplanata ROELOFS (Coleoptera: Curculionidae) (뽕나무애바구미의 생태 및 방제에 관한 연구)

  • 백현준;백운하
    • Journal of Sericultural and Entomological Science
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    • v.18 no.2
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    • pp.65-78
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    • 1976
  • The mulberry small weevil, Baris deplanata ROELOFS, has highly infested mulberry trees in Korea. As the damage caused by the mulberry small weevil in mulberry fields has been increased over the country since 1969, the authors has carried out a series of biological and controlling studies on the pest from 1971 to 1972. The results obtained are summarized as follows. 1. The adult weevil is elongate oval in shape with black in color and the probocis is long as usual in curculionidae. The size of adult female is 3.30${\pm}$0.04mm in length, 1.47${\pm}$0.04mm in width, and the length of proboscis is 1.25${\pm}$0.014mm, while adult male is 3.28${\pm}$0.06mm in length, 1.40${\pm}$0.04mm in width, and the length of proboscis is 1.30${\pm}$0.02mm. The antenna is geniculate consisting of 12 segments. The terminal sternite of the abdomen has a pointed tip in male but not in female. 2. The egg is long oval in shape, milky white in color, 0.51${\pm}$0.05mm in length and 0.32${\pm}$0.02mm in width. 3. The mature larva is cylindrical and light yellowow in color except the head of dark brown, and legless, 3.88${\pm}$0.06mm in length, 1.40${\pm}$0.02mm width, each segment bearing many wrinkless and short setae. 4. The pupa is long oval, milky white and exarate, 3.53${\pm}$0.09 in length, 1.40${\pm}$0.03mm in width. 5. Majority of the species has one generation through a year and overwinters as adult in xylem of withered branch and come out again from late April to early May in next year. But some of the female oviposit in the same year and the offsprings overwinter as larva (0.4%) or pupa (0.1%) 6. The eggs are mostly laid under the cork layer of withered branch and the number of eggs deposited by an adult female is 73.44${\pm}$8.74, the average egg-laying period is 33.88${\pm}$6.04 days. The incubation period is 11.69${\pm}$0.39 days, the larval period 45.04${\pm}$1.63 and the pupal period 11.05${\pm}$0.49 days. The period of adult's activity is 46.7${\pm}$5.90 days. 7. The larvae feed on the cambium under the bark and adults feed on the winter bud, the latent bud, the leaf stalk and the base of newly shoot. 8. An active period of adults was observed during the period of 4 months from April to July. However, the peak of adult-density occurred in the early May (in the fields of spring-prunning) and early to middle June(in the fields of summer-prunning). 9. There is a positive correlation between the density of larvae and diameter and length of the branches. 10. The pattern of distributions of the adult of mulberry small weevil is negative binomial distribution. 11. The chalcid fly was disclosed to be a natural enemy which was parasite on the larvae of mulberry small weevil and its parasitic ratio was 11.9%. 12. Phosvel D, Malix D, Salithion EC, DDVP EC, and Phosvel EC were effective for the control of adults and Satchukoto-S EC, and Salithio EC were effective for the control of larvae.

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Transformation of Adult Mesenchymal Stem Cells into Cardiomyocytes with 5-azacytidine: Isolated from the Adipose Tissues of Rat (성체 백서의 지방조직에서 추출한 중간엽 줄기세포의 5-azacytidine을 이용한 심근세포 분화 유도)

  • Choe Ju-Won;Kim Yong-In;Oh Tae-Yun;Cho Dai-Yoon;Sohn Dong-Suep;Lee Tae-Jin
    • Journal of Chest Surgery
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    • v.39 no.7 s.264
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    • pp.511-519
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    • 2006
  • Background: Loss of cardiomyocytes in the myocardial infarction leads to regional contractile dysfunction, and necrotized cardiomyocytes in infracted ventricular tissues are progressively replaced by fibroblasts forming scar tissue. Although cardiomyoplasty, or implantation of ventricular assist device or artificial heart was tried in refractory heart failure, the cardiac transplantation was the only therapeutic modality because these other therapeutic strategies were not permanent. Cell transplantation is tried instead of cardiac transplantation, especially bone marrow is the most popular donated organ. But because bone marrow aspiration procedure is invasive and painful, and it had the fewer amounts of cellular population, the adipose tissue is recommended for harvesting of mesenchymal stem cells. Material and Method: After adipose tissues were extracted from abdominal subcutaneous adipose tissue and intra-abdominal adipose tissue individually, the cellular components were obtained by same method. These cellular components were tried to transformation with the various titers of 5-azacytidine to descript the appropriate concentration of 5-azacytidine and possibility of transformation ability of adipose tissue. Group 1 is abdominal subcutaneous adipose tissue and Group 2 is intra-abdominal adipose tissue-retroperitoneal adipose tissue and omentum. Cellular components were extracted by collagenase and $NH_4Cl$ et al, and these components were cultured by non-induction media - DMEM media containing 10% FBS and inducted by none, $3{\mu}mol/L,\;6{\mu}mol/L,\;and\;9{\mu}mol/L$ 5-azacytidine after the 1st and 2nd subculture. After 4 weeks incubation, tile cell blocks were made, immunostaining was done with the antibodies of CD34, heavy myosin chain, troponin T, and SMA. Result: Immunostaining of the transformed cells for troponin T was positive in the $6{\mu}mol/L\;&\;9{\mu}mol/L$ 5-azacytidine of Group 1 & 2, but CD34 and heavy myosin chain antibodies were negative and SMA antibody was positive in the $3{\mu}mol/L\;&\;6{\mu}mol/L$ 5-azacytidne of Group 2. Conclusion: These observations confirm that adult mesenchymal stem cells isolated from the abdominal subcutaneous adipose tissues and intra-abdominal adipose tissues can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.

Pro-inflammatory Cytokine Expression Through NF-${\kappa}B/I{\kappa}B$ Pathway in Lung Epithelial Cells (폐 상피세포에서 NF-${\kappa}B/I{\kappa}B$ 경로에 의한 염증매개 사이토카인의 발현)

  • Park, Gye-Young;Lee, Seung-Hee;HwangBo, Bin;Yim, Jae-Joon;Lee, Choon-Taek;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo;Yoo, Chul-Gyu
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.3
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    • pp.332-342
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    • 2000
  • Background : The importance of pro-inflammatory cytokines, especially tumor necrosis factor $\alpha$ (INF-$\alpha$) and interleukin-1$\beta$ (IL-1$\beta$), have been extensively documented in the generation of inflammatory lung disease. Lung epithelial cells are also actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. Understanding the mechanism of pro-inflammatory cytokine expression in lung epithelial cells is crucial to the development of new therapeutic modalities for inflammatory lung disease. Transcription of most pro-inflammatory cytokines is dependent on the activation of NF-${\kappa}B$. However, the relationship between pro-inflammatory cytokine expression and NF-${\kappa}B/I{\kappa}B$ pathway in lung epithelial cells is not clear. Methods : BEAS-2B, A549, Na-H157, NCI-H719 cells were stimulated with IL-$1{\beta}$ or TNF-$\alpha$ at various times, and then IL-8 and TNF-$\alpha$mRNA expressions were assayed by Northern blot analysis. IL-$1{\beta}$ or TNF-$\alpha$-induced NF-${\kappa}B$ activation was assessed by the nuclear translocation of p65 NF-${\kappa}B$ subunit. The degradation of $I{\kappa}B{\alpha}$ and $I{\kappa}B{\beta}$ by IL-$1{\beta}$ or TNF-$\alpha$stimulation was assayed by Western blot analysis. The phosphorylation of $I{\kappa}B{\alpha}$ was evaluated by Western blot analysis after pre-treating cells with proteasome inhibitor followed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation. The basal level of IKK $\alpha$ expression was evaluated by Western blot analysis. Results: $I{\kappa}B{\alpha}$ and $I{\kappa}B{\alpha}$ was rapidly degraded after 5 minutes of incubation with IL-$1{\beta}$ or TNF-$\alpha$ in BEAS-2B, A549, and NCI-H157 cells. The activation of NF-${\kappa}B{\alpha}$ and the induction of IL-8 and TNF-$\alpha$ mRNA expression were observed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in these cells. In contrast, neither the changes in NF-${\kappa}B/I{\kappa}B$ pathway nor IL-8 and TNF-$\alpha$mRNA expression was induced by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in NCI-H719 cells. IL-$1{\beta}$ and TNF-$\alpha$-induced $I{\kappa}B$ phosphorylation was observed in BEAS-2B, A549, and NCI-H157 cells, but not in NCI-H719 cells. The basal level of IKK$\alpha$ expression was not different between cell. Conclusion : NF-${\kappa}B/I{\kappa}B$ pathway plays an important role in the expression of pro-inflammatory cytokine in most lung epithelial cells. The absence of the effect on NF-${\kappa}B/I{\kappa}B$ pathway in NCI-H719 cells sæms to be due to the defect in the intracellular signal transduction pathway upstream to IKK.

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Effect of Verapamil on Cellular Uptake of Tc-99m MIBI and Tetrofosmin on Several Cancer Cells (수종의 암세포에서 Verapamil이 Tc-99m MIBI와 Tetrofosmin의 섭취에 미치는 영향)

  • Kim, Dae-Hyun;Yoo, Jung-Ah;Suh, Myung-Rang;Bae, Jin-Ho;Jeong, Shin-Young;Ahn, Byeong-Cheol;Lee, Kyu-Bo;Lee, Jae-Tae
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.1
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    • pp.85-98
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    • 2004
  • Purpose: Cellular uptake of $^{99}mTc$-sestamibi (MIBI) and $^{99}mTc$-tetrofosmin (TF) is low in cancer cells expressing multidrug resistance(MDR) by p-glycoprotein(Pgp) or multidrug related protein(MRP). Verapamil is known to increase cellular uptake of MIBI in MDR cancer cells, but is recently reported to have different effects on tracer uptake in certain cancer cells. This study was prepared to evaluate effects of verapamil on cellular uptake of MIBI and TF in several cancer cells. Materials and Methods: Celluar uptakes of Tc-99m MIBI and TF were measured in erythroleukermia K562 cell, breast cancer MCF7 cell, and human ovarian cancer SK-OV-3 cells, and data were compared with those of doxorubicin-resistant K562(Ad) cells. RT-PCR and Western blot analysis were used for the detection of mdr1 mRNA and Pgp expression, and to observe changes in isotypes of PKC enzyme. Effects of verapamil on MIBI and TF uptake were evaluated at different concentrations upto $200{\mu}M\;at\;1{\times}10^6\;cells/ml\;at\;37^{\circ}C$. Radioactivity in supernatant and pellet was measured with gamma counter to calculate cellular uptake ratio. Toxicity of verapamil was measured with MTT assay. Results: Cellular uptakes of MIBI and TF were increased by time in four cancer cells studied. Co-incubation with verapamil resulted in an increase in uptake of MIBI and TF in K562(Adr) cell at a concentration of $100{\mu}M$ and the maximal increase at $50{\mu}M$ was 10-times to baseline. In contrast, uptakes of MIBI and TF in K562, MCF7, SK-OV3 cells were decreased with verapamil treatment at a concentration over $1{\mu}M$. With a concentration of $200{\mu}M$ verapamil, MIBI and TF uptakes un K562 cells were decreased to 1.5 % and 2.7% of those without verapamil, respectively. Cellular uptakes of MIBI and TF in MCF7 and SK-OV-3 cells were not changed with $10{\mu}M$, but were also decreased with verapamil higher than $10{\mu}M$, resulting 40% and 5% of baseline at $50{\mu}M$. MTT assay of four cells revealed that K562, MCF7, SK-OV3 were not damaged with verapamil at $200{\mu}M$. Conclusion: Although verapamil increases uptake of MIBI and TF in MDR cancer cells, cellular uptakes were further decreased with verapamil in certain cancer cells, which is not related to cytotoxicity of drug. These results suggest that cellular uptakes of both tracers might differ among different cells, and interpretation of changes in tracer uptake with verapamil in vitro should be different when different cell lines are used.

A Novel in Vitro Method for the Metabolism Studies of Radiotracers Using Mouse Liver S9 Fraction (생쥐 간 S9 분획을 이용한 방사성추적자 대사물질의 새로운 체외 측정방법)

  • Ryu, Eun-Kyoung;Choe, Yearn-Seong;Kim, Dong-Hyun;Lee, Sang-Yoon;Choi, Yong;Lee, Kyung-Han;Kim, Byung-Tae
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.4
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    • pp.325-329
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    • 2004
  • Purpose: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of $^{18}F$-labeled radiotracers. Materials and Methods: Mouse liver S9 fraction was isolated at au early step in the course of microsome preparation. The in vitro metabolism studies were tarried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at $37^{\ciirc}C$, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. Results: The radiotracer $[^{18}F]1$ underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer $[^{18}F]2$ was metabolized to three metabolites including $4-[^{18}F]fluorobenzoic$ acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. Conclusion: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.

Removal of ${\alpha}$-Gal Epitopes in Aortic Valve and Pericardium of Pig Using Green Coffee Bean ${\alpha}$-Galactosidase (돼지의 대동맥 판막 및 심낭에서 녹색콩 알파-갈락토시다아제를 이용한 알파-갈 항원결정인자 제거)

  • Park, Seong-Sik;Kim, Woong-Han;Kim, Kyung-Hwan;Lee, Chang-Ha;Choi, Sun-Young;Lee, Cheul;Oh, Sam-Sae;Kim, Kwan-Chang;Kim, Yong-Jin
    • Journal of Chest Surgery
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    • v.41 no.1
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    • pp.12-24
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    • 2008
  • Background: It is currently thought that tissue valve degeneration is related to an animal's immune response, which is mainly due to cell surface ${\alpha}$-Gal epitopes. Cell surface ${\alpha}$-Gal epitopes are known to be degraded by the enzyme called green coffee bean ${\alpha}$-Galactosidase. It is also well known that ${\alpha}$-Gal epitopes are immunologically stained by Griffonia Simplicifolia isolectin type B4. We know that many commercially available tissue valves are made of aortic valves and pericardial tissue of pig. So, we investigated whether ${\alpha}$-Gal epitopes of the aortic valve and pericardial tissue of a pig can be removed by green coffee bean ${\alpha}$-Galactosidase, and we did so by comparing immunologic staining of the tissues before and after the enzyme treatment. Material and method: After treating fresh porcine aortic valve and pericardial tissue with green coffee bean ${\alpha}$-Galactosidase at concentrations of 0.5 unit/mL, 1.0 unit/mL, 2.0 unit/mL, respectively, under the condition of pH 6.5, temperature. $4^{\circ}C$ and 24 hours of incubation, each sample was stained with Griffonia Simplicifolia isolectin type B4 immunpfluorescent labeling. We then examined whether the ${\alpha}$-Gal epitopes were reduced or abolished in each consecutive. concentration of green coffee bean ${\alpha}$-Galactosidase by comparing the degree of the Griffonia Simplicifolia isolectin B4 staining in each sample. Result: In the pig aortic valve tissue, a 1.0 unit/mL concentration of green coffee bean ${\alpha}$-Galactosidase at pH 6.5, $4^{\circ}C$ and reaction for 24 hours was enough for complete removal of ${\alpha}$-Gal epitopes from the cell sur face on the immunostaining with Griffonia Simplicifolia isolectin B4. On the other hand, more ${\alpha}$-Gal epitopes were present in the pig pericardial tissue on Griffonia Simplicifolia isolectin B4 staining before the enzyme treatment, and 1.0 unit/mL of galactosidase was not sufficient for complete removal of ${\alpha}$-Gal from the tissue. 2.0 units/mL of green coffee bean ${\alpha}$-Galactosidase was needed to completely remove the ${\alpha}$-Gal epitopes from the pericardial tissue on immunostaining. Conclusion: The ${\alpha}$-Gal epitopes of the pig's aortic valve and pericardial tissue were successfully stained with Griffonia Simplicifolia isolectin B4. We could remove nearly all the ${\alpha}$-Gal epitopes using green coffee bean ${\alpha}$-Galactosidase at the concentration of 1.0 unit/mL in the aortic valve. Of pig, and 2.0 unit/mL was need to nearly completely remove all the ${\alpha}$-Gal epitopes in the pericardial tissue of pig under the condition of pH 6.5, $4^{\circ}C$ and 24 hours of reaction time. In the near future, removal of ${\alpha}$-Gal epitapes in the pig's aortic valve and pericardial tissue will become a powerful tool for the improvement of the tissue valve durability. It needs to be determined if ${\alpha}$-galactosidase treated pig tissue is immune to human anti-Gal antibody or anit-Gal mooclonal antibodies.