• The Korean Journal of Nuclear Medicine
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• v.39 no.1
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• pp.34-43
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• 2005

Purpose: $^{99m}Tc$-sestamibi(MIBI) and $^{99m}Tc$-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of $^{99m}Tc$-MIBI and $^{99m}Tc$-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. Materials and Methods: HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. Results: RT-PCR, western blot analysis of the cells and irnrnunochemical staining revealed selective expression of Pgp and MRP for HCY15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10- and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells (p<0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 240 min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to 240 min with CsA. But increases in tumoral uptake were not significantly different between MIBI and tetrofosmin for both tumors. Conclusion: MIBI seems to be a better tracer than tetrofosmin for evaluating MDR reversal effect of the modulators in vitro, but these differences were not evident in vivo tumoral uptake. Both MIBI and tetrofosmin seem to be suitable tracers for imaging Pgp- and MRP-mediated drug resistance in tumors.

• The Korea Journal of Herbology
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• v.23 no.2
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• pp.87-97
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• 2008

Objectives : The present study was carried out to investigate the effect of Herba Polygalae Japonica on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dessiciated with collagenase (1 ${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with extract of Herba Polygalae Japonica (EPJ), incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometer, ELISA, RT-PCR and immunocytochemistry stain. Results : A significant cytotoxicity by drug treatment was not observed. The cell number ratio of granulocyte, CD3e-/CCR3+, CD3e+CD69+, CD4+, CD23+/B220+ cells was increased in rmIL-5/rIL-3 treated control group compared to the normal group. Cells numbers in the experimental animal group treated with EPJ was all decreased. In ELISA analysis, IL-4, IL-5, IL-13 levels and histamine release level were increased in the control group compared to the normal animal group, then significantly decreased in the experimental group with 100 ${\mu}g$/ml of EPJ treatment. In RT-PCR analysis, mRNA expressions of IL-4, IL-5, IL-13, CCR3 and eotaxin were increased in the control group compared to the normal animal group, then decreased in the experimental group with 100 ${\mu}g$/ml of EPJ treatment. And eosinophil proliferation levels were 18847${\pm}$,1527 (cpm) in the control group, 4676${\pm}$972 (cpm) in the positive control group, and 7709 ${\pm}$ 549 (cpm), 16839 ${\pm}$ 1403 (cpm), 16385 ${\pm}$ 1723 (cpm) in the experimental group with 100 ${\mu}g$/ml, 10 ${\mu}g$/ml, 1 ${\mu}g$/ml of EPJ treatment. Conclusions : The present data suggested that Herba polygalae japonica may have an effects on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of EPJ.

• The Journal of Pediatrics of Korean Medicine
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• v.32 no.3
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• pp.1-15
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• 2018

Objectives Alismatis Rhizoma has been known to suppress inflammation and allergic reaction. However, the cellular target of Alismatis Rhizoma and its mechanism of action remain unclear. This study was designed to examine the effect of Alismatis Rhizoma extract (ALC) on the RBL-2H3 mast cells in vitro and on the OVA/alum sensitized mice ex vivo. Methods In the study, RBL-2H3 mast cells were cultured in minimal essential medium (MEM) for 24 hours, and treated separately with cyclosporin A and varying doses of ALC, and then stimulated with Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and Ionomycin ($0.5{\mu}M$). The levels of IL-13, IL-4 were measured by ELISA analysis. The mRNA levels of IL-4, IL-5, IL-6, IL-13, GM-CSF, $TNF-{\alpha}$ were analyzed with Real-time PCR. Also, manifestations of MAPKs transcription factors and $NF-{\kappa}B$ p65 translocation were analyzed by western blotting in vitro. Subsequently, for ex vivo experiment, we induced allergic inflammation on Balb/c mice by OVA/alum and administered ALC orally. And we measured serum OVA-specific IgE level and IL-4, IL-13 in the splenocyte culture supernatant by ELISA analysis. Results ALC was shown to suppress mRNA expression of IL-4, IL-5, IL-6, IL-13, GM-CSF, $TNF-{\alpha}$, and to inhibit the IL-13, IL-4 production. Also ALC reduced an activation of mast cells specific signal MAPKs transcription factors and $NF-{\kappa}B$ p65 from the western blot analysis in in vitro experiment. In ex vivo, ALC oral adminstration decreased the level of OVA-specific IgE in serum, and IL-4, IL-13 in the splenocyte culture supernatant. Conclusions ALC is shown to reduce inflammation and allergic response by suppressing Th2 cytokines through the regulation of transcription factors MAPKs and $NF-{\kappa}B$ p65 in mast cells. Administration of ALC suppressed OVA-specific IgE in ovalbumin allergy model through the inhibition of Th2 cytokine. In conclusion, ALC can be considered as an effective treatment for allergic diseases such as atopic dermatitis.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.331-337
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• 2009

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a well-known antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

• Proceedings of the Korean Information Science Society Conference
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• 2007.06b
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• pp.273-278
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• 2007

검사점 및 복구 도구(Checkpointing & Recovery Facility)를 이용하여 임베디드 시스템에서 결함 허용(Fault Tolerance) 기법을 적용할 경우 쓰기 작업의 오버헤드로 인해 실용성이 크게 떨어지게 된다. 실시간 운영체제와 함께 어떠한 한계 상황에서 결함 허용 및 복구 도구가 오히려 시스템의 성능을 저하시키는 요인으로 작용하게 되면 이는 결국 쓸모없는 도구가 되어 사용되지 않을 것이다. 따라서 프로세스의 복구를 위해 저장하는 프로세스 이미지의 기록에 소요되는 시간을 크게 낮추어야만 비로소 검사점 도구가 그 진가를 발휘하게 될 수 있다. 본 논문에서는 NVSRAM(Non Volatile SRAM)을 검사점 및 복구 도구의 저장 장치로 활용함으로써 기존의 검사점 도구에서 성능을 저하시키는 주원인이었던 검사점 기록의 오버헤드를 개선하기 위한 연구를 수행하였다. 검사점 기록 시간을 줄이기 위한 방법으로 주 메모리에 저장된 프로세스의 복구와 관련된 데이터를 SRAM 특성을 갖는 비휘발성 저장 장치인 NVSRAM에 저장하여 디스크 접근에 소요되는 시간을 최소화시킴으로써 임베디드 시스템에서 실용적으로 사용 가능한 검사점 도구를 구현하였고, 이러한 연구의 결과를 검증하기 위해 기존 시스템에서 저장 장치로 사용되던 플래시 메모리, 주 메모리, 원격 메모리를 사용하는 경우의 성능과 NVSRAM을 활용할 때의 성능을 비교해 보았다. 본 연구에서 제안하는 결함 허용 도구는 실제 시스템에 적용하여 효과적인 성능을 발휘할 수 있을 것이며, 차세대 메모리를 이용한 결함 허용 도구의 연구에 기여를 할 수 있을 것으로 기대된다.ate첨가배지(添加培地)에서 가장 저조(低調)하였다. vitamin중(中)에서는 niacin과 thiamine첨가배지(添加培地)에서 근소(僅少)한 증가(增加)를 나타내었다.소시켜 항이뇨 및 Na 배설 감소를 초래하는 작용과, 둘째는 신경 경로를 통하지 않고, 아마도 humoral factor를 통하여 신세뇨관에서 Na 재흡수를 억제하는 작용이 복합적으로 나타내는 것을 알 수 있었다.으로 초래되는 복합적인 기전으로 추정되었다., 소형과와 기형과는 S-3에서 많이 나왔다. 이상 연구결과에서 입도분포가 1.2-5mm인 것이 바람직한 것으로 나타났다.omopolysaccharides로 확인되었다. EPS 생성량이 가장 좋은 Leu. kimchii GJ2의 평균 분자량은 360,606 Da이었으며, 나머지 두 균주에 대해서는 생성 EPS 형태와 점도의 차이로 미루어 보아 생성 EPS의 분자구조와 분자량이 서로 다른 것으로 판단하였다.TEX>개로 통계학적으로 유의한 차이가 없었다. Heat shock protein-70 (HSP70)과 neuronal nitric oxide synthase (nNOS)에 대한 면역조직화학검사에서 실험군 Cs2군의 신경세포가 대조군 12군에 비해 HSP70과 nNOS의 과발현을 보였으며, 이는 통계학적으로 유의한 차이를 보였다(p<0.05). nNOS와 HSP70의 발현은 강한 연관성을 보였고(상관계수 0.91, p=0.000), nNOS를 발현하는 세포가 동시에 HSP70도 발현함을 확인할 수 있었다. 결론: 우리는 cyclosporin A가 토끼의 25분간의 척수허혈에 대해 척수보호 효과가 있었으며 이는 HSP70의

• International Journal of Oral Biology
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• v.33 no.4
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• pp.205-211
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• 2008

Gingival overgrowth can cause dental occlusion and seriously interfere with mastication, speech, and dental hygiene. It is observed in 25 to 81% of renal transplant patients treated with cyclosporine A (CsA). CsA-induced gingival overgrowth (CIGO) is caused by quantitative alteration of the extracellular matrix components, particularly collagen. However, the molecular mechanisms involved in the pathogenesis of CIGO remain poorly understood, despite intense clinical and laboratory investigations. The aim of the present work is to identify differentially expressed genes closely associated with CIGO. Human gingival fibroblasts were isolated by primary explant culture of gingival tissues from five healthy subjects (HGFs) and two patients with the CIGO (CIGO-HGFs). The proliferative activity of CsA-treated HGFs and CIGO-HGFs was examined using the MTT assay. The identification of differentially expressed genes in CsA-treated CIGO-HGF was performed by differential display reverse transcriptase-polymerase chain reaction (RT-PCR) followed by DNA sequencing. CsA significantly increased the proliferation of two HGFs and two CIGO-HGFs, whereas three HGFs were not affected. Seven genes, including the beta subunit of prolyl 4-hydroxylase (P4HB) and testican 1, were upregulated by CsA in a highly proliferative CIGO-HGF. The increased P4HB and testican-1 mRNA levels were confirmed in CsA-treated CIGO-HGFs by semiquantitative RT-PCR. Furthermore, CsA increased type I collagen mRNA levels and suppressed MMP-2 mRNA levels, which are regulated by P4HB and testican-1, respectively. These results suggest that CsA may induce gingival overgrowth through the upregulation of P4HB and testican-1, resulting in the accumulation of extracellular matrix components.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.3
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• pp.590-598
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• 2009

The purpose of this research is to evaluate the effect of Mahaenggamseok-tang-gagambang (MGTG) on airway hyper- responsiveness (AHR), immune cells, cytokines and lung tissue in OVA-induced asthmatic mice. C578L/6 mice were injected, inhaled and sprayed with OVA for 12 weeks (3times a week) for asthma sensitization and challenge. Two experimental groups were treated with different concentrations of MGTG (400 mg/kg and 200 mg/kg) extract and cyclosporin A (10 mg/kg) for the later 8 weeks. Enhanced pause (Penh) levels were measured by whole body plethysmography. Immune cells were analyzed by flow cytometer in peripheral blood monocyte cell (PBMC) and lung cells. The IL-1b, IL-12, IFN-${\gamma}$, OVA-lgE, IL-4, IL-5, TNF-${\alpha}$ were analyzed by ELISA kit in serum and splenocyte+a-cCDS/a-CD28. Enhanced pause (Penh) levels of the MGTG groups (400 mg/kg and 200 mg/kg) were decreased significantly compared with that of control group. The numbers of MGTG groups (400 mg/kg and 200 mg/kg) on lung total cells were decreased significantly compared with that of control group. The numbers of MGTG groups (400 mg/kg and 200 mg/kg) on $CD3^+/CD69^+$, $B220^+/CD22^+$, $B220^+/CD23^+$, $B220^+/lgE^+$, $CCR3^+$ cells were decreased significantly compared with that of control group. The number of MGTG group (400 mg/kg) on $CD3^+/CD49b^+$ cells was decreased significantly compared with that of control group. The level of MGTG groups (400 mg/kg and 200 mg/kg) on IL-4, IL-5, IL-12, TNF-${\alpha}$, OVA-lgE were decreased significantly compared with that of control group. The level of MGTG group (400 mg/kg) on IL-1b, IL-1S, OVA-lgE were decreased significantly compared with that of control group. These results demonstrate that MGTG could be a desirable alternative therapy for allergic asthma by inhibiting the expression of immune cells, the activation of inflammatory mediator.

• Archives of Pharmacal Research
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• v.24 no.2
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• Molecules and Cells
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• v.38 no.12
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• pp.1064-1070
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• 2015

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10-100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO ($0.1-0.5{\mu}m$), a specific mitochondrial antioxidants, and cyclosporin A ($1-5{\mu}m$), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam ($1-50{\mu}m$), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.

• Korean Journal of Clinical Pharmacy
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• v.20 no.1
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• pp.9-16
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• 2010

Purpose: 본 연구는 한국인 성인 조혈모세포이식환자를 대상으로 경구용 사이클로스포린의 집단약동학 분석을 통하여 사이클로스포린의 약동학적 파라미터에 영향을 미치는 요인 분석을 실시하고자 하였다. Methods: 2000년 12월부터 2006년 8월까지 서울대학교병원에서 동종조혈모세포이식을 받고 면역억제제로 사이클로스포린을 복용한 성인 백혈병환자를 대상으로 후향적으로 자료를 수집하였다. 사이클로스포린의 약동학에 영향을 미치는 인자로는 연령, 성별, 이식 후 날짜, 신기능, 공여자와의 관계, 질병의 종류, 혈중 빌리루빈 농도, 사이클로스포린의 대사를 유도하는 프레드니솔론의 투여량, 헤마토크리트, 사이클로스포린의 대사를 저해하는 약물의 병용여부 등을 검토하였다. 분석은 NONMEM$^{(R)}$ VI 프로그램을 이용하였으며, 변수를 추가하지 않은 기본 모형을 만든 후에 단계적인 요인의 추가와 제거를 통해 최종모형을 제작하였다. Results: 최종 상관 모형은 다음과 같다; CL/F (L/h) = $85.6{\times}e^{(0.646\;{\times}\;HCT/28.9\;+\;0.0464\;{\times}\;Gender)}$. 사이클로스포린의 겉보기 클리어런스는 환자의 성별이 남자일 때 또는 헤마토크릿이 감소할수록 증가하였다. 그 외 파라미터는 다음과 같이 계산되었다; $K_{\alpha}=0.0787\;(h^{-1})$; Q=57.1(L/kg/h); $V_{d-central\;compartment}$=1,100 (L); $V_{d-peripheral\;compartment}$ = 213,000(L). 개체간 편차는 40% 미만이었으며, 개체내 편차를 포함하는 잔차는 24.02%였다. Conclusions: 사이클로스포린의 약동학적 특징과 그 클리어런스에 영향을 끼칠 수 있는 임상적 요인을 이해하는 것은 환자 개개인의 용량과 용법의 결정 및 이상반응 발생의 예방에 유용할 수 있다. 한국인 조혈모세포이식환자에서 사이클로스포린의 약동학에 영향을 미치는 최종 파라미터를 구한 본 연구의 결과는 조혈모세포이식을 받은 한국인 성인환자에서 사이클로스포린의 모니터링 및 용량조절에 유용할 것으로 전망된다.