• 제목/요약/키워드: Kinase

검색결과 4,541건 처리시간 0.033초

The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species

  • Joo, Hee Kyoung;Lee, Yu Ran;Kang, Gun;Choi, Sunga;Kim, Cuk-Seong;Ryoo, Sungwoo;Park, Jin Bong;Jeon, Byeong Hwa
    • Molecules and Cells
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    • 제38권12호
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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.

임상화학검사실에서 회수율 실험의 실증적 연구 (An Empirical Study of the Recovery Experiment in Clinical Chemistry)

  • 장상우;이상곤;송은영;박용원;박병옥
    • 대한임상검사과학회지
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    • 제38권3호
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    • pp.184-188
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    • 2006
  • The purpose of the recovery experiment in clinical chemistry is performed to estimate proportional systematic error. We must know all measurements have some error margin in measuring analytical performance. Proportional systematic error is the type of error whose magnitude increases as the concentration of analyte increases. This error is often caused by a substance in the sample matrix that reacts with the sought for analyte and therefore competes with the analytical reagent. Recovery experiments, therefore, are used rather selectively and do not have a high priority when another analytical method is available for comparison purposes. They may still be useful to help understand the nature of any bias revealed in the comparison of kit experiments. Recovery should be expressed as a percentage because the experimental objective is to estimate proportional systematic error, which is a percentage type of error. Good recovery is 100.0%. The difference between 100 and the observed recovery(in percent) is the proportional systematic error. We calculated the amount of analyte added by multiplying the concentration of the analyte added solution by the dilution factor(mL standard)/(mL standard + mL specimen) and took the difference between the sample with addition and the sample with dilution. When making judgments on method performance, the observed that the errors should be compared to the defined allowable error. The average recovery needs to be converted to proportional error(100%/Recovery) and then compared to an analytical quality requirement expressed in percent. The results of recovery experiments were total protein(101.4%), albumin(97.4%), total bilirubin(104%), alkaline phosphatase(89.1%), aspartate aminotransferase(102.8), alanine aminotransferase(103.2), gamma glutamyl transpeptidase(97.6%), creatine kinase(105.4%), lactate dehydrogenase(95.9%), creatinine(103.1%), blood urea nitrogen(102.9%), uric acid(106.4%), total cholesterol(108.5), triglycerides(89.6%), glucose(93%), amylase(109.8), calcium(102.8), inorganic phosphorus(106.3%). We then compared the observed error to the amount of error allowable for the test. There were no items beyond the CLIA criterion for acceptable performance.

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Effects of Insulin and IGFs on Phosphate Uptake in Primary Cultured Rabbit Renal Proximal Tubule Cells

  • Han, Ho-Jae;Park, Kwon-Moo
    • The Korean Journal of Physiology
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    • 제30권1호
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    • pp.63-76
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    • 1996
  • The aim of present study was to characterize phosphate uptake and to investigate the mechanism for the insulin and insulin-like growth factor(IGF) stimulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells. Results were as follows : 1. The primary cultured proximal tubule cells had accumulated $6.68{\pm}0.70$ nmole phosphate/mg protein in the presence of 140 mM NaCl and $2.07{\pm}0.17$ nmole phosphate/mg protein in the presence of 140 mM KCl during a 60 minute uptake period. Raising the concentration of extracellular phosphate to 100 mM$(48.33{\pm}1.76\;pmole/mg\;protein/min)$ induced decrease in phosphate uptake compared with that in control cells maintained in 1 mM phosphate$(190.66{\pm}13.01\;pmole/mg\;protein/min)$. Optimal phosphate uptake was observed at pH 6.5 in the presence of 140 mM NaCl. Phosphate uptake at pH 7.2 and pH 7.9 decreased to $83.06{\pm}5.75%\;and\;74.61{\pm}3.29%$ of that of pH 6.5, respectively. 2. Phosphate uptake was inhibited by iodoacetic acid(IAA) or valinomycin treatment $(62.41{\pm}4.40%\;and\;12.80{\pm}1.64%\;of\;that\;of\;control,\;respectively)$. When IAA and valinomycin were added together, phosphate uptake was inhibited to $8.04{\pm}0.61%$ of that of control. Phosphate uptake by the primary proximal tubule cells was significantly reduced by ouabain treatment$(80.27{\pm}6.96%\;of\;that\;of\;control)$. Inhibition of protein and/or RNA synthesis by either cycloheximide or actinomycin D markedly attenuated phosphate uptake. 3. Extracellular CAMP and phorbol 12-myristate 13 acetate(PMA) decreased phosphate uptake in a dose-dependent manner in all experimental conditions. Treatment of cells with pertussis toxin or cholera toxin inhibited phosphate uptake. cAMP concentration between $10^{-6}\;M\;and\;10^{-4}\;M$ significantly inhibited phosphate uptake. Phosphate uptake was blocked to about 25% of that of control at 100 ng/ml PMA. 3-Isobutyl-1-methyl-xanthine(IBMX) inhibited phosphate uptake. However, in the presence of IBMX, the inhibitory effect of exogenous cAMP was not significantly potentiated. Forskolin decreased phosphate transport. Acetylsalicylic acid did not inhibit phosphate uptake. The 1,2-dioctanoyl-sn-glycorol(DAG) and 1-oleoyl-2-acetyl-sn- glycerol(OAG) showed a inhibitory effect. However, staurosporine had no effect on phosphate uptake. When PMA and staurosporine were treated together, inhibition of phosphate uptake was not observed. In conclusion, phosphate uptake is stimulated by high sodium and low phosphate and pH 6.5 in the culture medium. Membrane potential and intracellular energy levels are also an important factor fer phosphate transport. Insulin and IGF-I stimulate phosphate uptake through a mechanisms that involve do novo protein and/or RNA synthesis and decrease of intracellular cAMP level. Also protein kinase C(PKC) is may play a regulatory role in transducing the insulin and IGF-I signal for phosphate transport in primary cultured proximal tubule cells.

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EGFR 돌연변이 검출에 있어 PNA-Mediated Real-Time PCR Clamping과 직접 염기서열 분석법의 비교 분석 (Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection)

  • 김희정;김완섭;신경철;이관호;김미진;이정은;송규상;김선영;이계영
    • Tuberculosis and Respiratory Diseases
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    • 제70권1호
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    • pp.21-27
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    • 2011
  • Background: Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of $EGFR$ mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to $EGFR$ mutation status using both methodologies. Methods: Clinical specimens from 112 NSCLC patients were analyzed for $EGFR$ mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to $EGFR$-TKIs were compared. Results: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). Conclusion: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in $EGFR$ gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of $EGFR$ mutations in clinical setting.

적소두 추출물이 톨유사수용체 활성 및 염증유발 사이토카인의 생성에 미치는 영향 (Effect of Vigna angularis on Toll-like Receptor Activation and Pro-inflammatory Cytokine Production)

  • 김미화;정시화;이승웅;김현규;박찬선;전병훈;오현미;노문철
    • 동의생리병리학회지
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    • 제26권4호
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    • pp.511-518
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    • 2012
  • The mechanisms of Toll-like receptor (TLR) signaling have been the focus of extensive studies because TLRs are the target of therapeutic intervention on multiple diseases. In this study, we investigated the inhibitory potential of Vigna angularis (azuki bean) on the TLR signaling. The effect of Vigna angularis extract (JSD) on TLR activation was investigated by assessing NF-${\kappa}B$ and AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) activity. JSD significantly inhibited SEAP activity induced by poly I:C (TLR3 ligand) and poly I (TLR7 ligand) in a dose-dependent manner at concentration below 100 ${\mu}g/ml$ with no sign of cytotoxicity. Pretreatment of JSD markedly suppressed mRNA expressions of pro-inflammatory cytokines and adhesive molecules such as TNF-${\alpha}$, IL-6, RANTES, MCP-1 and ICAM-1 induced by TLR ligands. It also diminished the phosphorylation of $I{\kappa}B$ kinase and $I{\kappa}B$, and followed by $I{\kappa}B$-mediated nuclear translocation of p50, p65, and phosphorylation of p38, JNK, and IRF signaling pathway. In conclusion, our results suggest that Vigna angularis has inhibitory activity on TLR-3 and -7 signaling and it can be further developed as a remedy in curing TLR-related multiple diseases.

인체폐암세포 NCI-H460 및 A549의 증식에 미치는 삼기보폐탕의 영향 비교 (Induction of Apoptosis by Samgibopae-tang in Human Non-small-cell Lung Cancer Cells)

  • 허만규;박철;최영현;감철우;박동일
    • 동의생리병리학회지
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    • 제21권4호
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    • pp.973-981
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    • 2007
  • In the present study, we investigated the antiproliferative activity of the water extract of Samgibopae-tang (SGBPT) in NCI-H460 and A549 non-small-cell lung cancer cell lines. We found that exposure of A549 cells to SGBPT resulted in the growth inhibition in a dose-dependent manner as measured by MTT assay, however SGBPT did not affect the growth of NCI-H460 cells. The antiproliferative effect by SGBPT treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. SGBPT treatment did not induce the cell cycle arrest in both cell lines, however the frequency of sub-G1 population was concentration-dependently increased by SGBPT treatment in A549 cells. SGBPT treatment partially induced the expression of tumor suppressor p53 in A549 cells and the expression of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was markedly increased in both transcriptional and translational levels in A549 cells. The up-regulation of p21 by SGBPT occurred in a similar a concentration dependent manner to that observed with the inhibition of cell viability and induction of sub-G1 population of the cell cycle. However SGBPT treatment did not affect other growth regulation-related genes such as early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), inducible nitric oxide synthease (iNOS), cyclooxygenases (COXs), telomere-regulatory factors in A549 as well as NCI-H460 cells. Taken together, these findings suggested that SGBPT-induced inhibition of human lung carcinoma A549 cell growth was aoosciated with the induction of p21 and the results provided important new insights into the possible molecular mechanisms of the anti-cancer activity of SGBPT.

RBL-2H3 세포에 있어서 꾸지뽕 당단백질에 의한 히스타민 방출 및 COX-2 활성 억제 효과 (Inhibitory Effect of Glycoprotein Isolated from Cudrania tricuspidata Bureau on Histamine Release and COX-2 Activity in RBL-2H3 Cells)

  • 오필선;이혜진;임계택
    • 한국식품과학회지
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    • 제41권4호
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    • pp.405-412
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    • 2009
  • 본 연구는 꾸지뽕 나무 열매로부터 75 kDa의 당단백질(꾸지뽕 당단백질)을 추출한 후 꾸지뽕 당단백질의 첨가에 따른 알레르기성 염증 인자인 histamine 유리 억제능력 및 COX-2의 활성 억제 효과를 평가하였다. RBL-2H3세포를 22시간 동안 IgE로 감작시킨 후, HSA를 처리하여 histamine의 유리양을 측정한 결과 꾸지뽕 당단백질을 처리한 농도가 증가함에 따라 histamine의 유리와 COX-2의 활성 억제율은 증가하였다. 또한 꾸지뽕 당단백질의 처리는 HSA에 의해 유도된 세포내 ROS 생성량을 농도에 의존적으로 억제하였다. 한편 꾸지뽕 당단백질을 농도별로 처리하여 세포내 단백질을 추출하여 western blot을 실시한 결과 100 ${\mu}g$/mL 농도의 꾸지뽕 당단백질을 처리한 그룹에서 ERK1/2, AP-1과 COX-2의 활성 수준은 현저히 억제 되었다(p<0.05). 따라서 이러 한 결과에 미루어볼 때, 꾸지뽕 당단백질은 세포내 해독효소의 활성을 증가시킴으로써 ROS 수준을 감소시켰으므로 꾸지뽕 당단백질의 역할이 다른 천연물 유래의 당단백질과 마찬가지로 특이적인 항산화 능력을 지니고 있음을 나타내며 histamine의 유리 억제와 COX-2의 활성이 억제되었을 것으로 생각된다. 이는 꾸지뽕 당단백질이 항 알레르기 효능을 갖는 물질로써 알레르기성 비염, 아토피 등과 같은 알레르기 관련 질환의 예방 및 치료제로 사용될 수 있을 것 사료된다.

정신분열증 치료제에 의한 사람 글루탐산염 탈수소효소 동종효소의 억제효과 (Inhibitory Effects of Human Glutamate Dehydrogenase Isozymes by Antipsychotic Drugs for Schizophrenia)

  • 남아름;김인식;양승주
    • 한국산학기술학회논문지
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    • 제17권1호
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    • pp.152-158
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    • 2016
  • 글루탐산염(Glutamate)은 척추동물의 중추신경계에서 중요한 흥분성 신경전달물질 중의 하나이다. 글루탐산염의 대사를 조절하는 사람 글루탐산염 탈수소 효소(hGDH)는 정신분열증(schizophrenia) 환자의 대뇌에서 발현이 증가한다는 연구들이 있었다. 본 연구에서는 정신분열증과 연관된 항정신성약물인 haloperidol, risperidone, (${\pm}$)-sulpride, chlopromazine hydrochloride, melperone, (${\pm}$)butaclamol, domperidone, clozapine에 의한 hGDH의 효소활성변화를 확인하고자 하였다. 우선, 유전자 재조합을 통해 hGDH 동종효소 hGDH1, hGDH2를 합성하였다. 합성된 hGDH1과 hGDH2에 대한 항정신성약물의 억제효과를 효소검사법(enzyme assay)을 통해 확인한 결과, haloperidol, (${\pm}$)-sulpride, melperone, clozapine에 의해 hGDH1과 hGDH2의 효소활성이 억제되었다. 또한, 단백질 인산화 효소 측정법(kinase assay)을 하여 haloperidol이 기질인 알파-케토글루타르산에 대하여는 비경쟁적 저해반응(noncompetitive inhibition)을, NADH에 대하여서는 반경쟁적 저해반응(uncompetitive inhibition)이 나타는 것을 확인하였다. 입체성 다른 자리 작동체(allosteric effector)인 L-leucine이 다른 정신병치료제에서는 hGDH2의 억제를 회복시켰지만 오직 haloperidol에서는 효소의 활성이 회복되지 않았다. 따라서 본 연구는 hGDH1과 hGDH2 에서 항정신성약물에 의한 효소활성 억제를 비교하여 확인하였으며, 중추신경계에서 haloperidol이 GDH 활성 조절과 함께 글루탐산 농도를 조절할 수 있다는 가능성을 제시한다.

원심성 운동 전 진동 훈련이 하퇴근육의 지연성 근통증에 미치는 영향 (The Effect of Vibration Training Pre-eccentric Exercise on Delayed Onset Muscle Soreness of Triceps Surae)

  • 김은숙;김미화;조유미;이완희
    • 한국산학기술학회논문지
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    • 제12권12호
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    • pp.5789-5796
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    • 2011
  • 본 연구는 왼쪽 하퇴삼두근의 원심성 운동으로 인한 지연성 근통증에서 진동훈련의 효과를 알아보고자 하였다. 이전에 규칙적인 하지 운동에 참여하지 않은 21명의 신체 건강한 남녀를 대상으로 진동훈련군과 대조군으로 나누었다. 통증척도, 발목 족저굴근의 근력, 하퇴삼두근의 둘레, 혈중 CK 수치를 원심성 운동전, 24시간, 48시간, 72시간 후에 측정하였다. 통증척도는 지연성 근통증 유발 후 48시 시점에 집단 간 유의한 차이를 보였고, 측정시기에 따라 집단 내 유의한 차이를 보였다(p<.05). 족저굴근의 근력은 진동훈련 집단 간의 유의한 차이는 보이지 않았으나, 측정시기에 따라 집단 내 유의한 차이를 보였다(p<.05). 하퇴삼두근 둘레는 진동훈련 집단 간 유의한 차이는 보이지 않았으나, 측정시기에 따라 집단 내 유의한 차이를 보였다(p<.05). 혈중 CK 수치는 진동훈련 집단 간에 유의한 차이는 보이지 않았으나, 측정시기에 따라 집단 내 유의한 차이를 보였다(p<.05). 본 연구결과로 원심성 운동 전 진동훈련은 통증을 억제하는 효과가 있으므로 익숙하지 않은 활동을 하거나, 스포츠 활동에서의 지연성 근통증을 예방하기 위한 한 방법으로 제시될 수 있을 것으로 사료된다.

Hepatic Steatosis Alleviated in Diabetic Mice upon Dietary Exposure to Fibroin via Transgenic Rice: Potential STAMP2 Involvement in Hepatocytes

  • Park, Ji-Eun;Jeong, Yeon Jae;Kim, Hye Young;Yoo, Young Hyun;Lee, Kwang Sik;Yang, Won Tae;Kim, Doh Hoon;Kim, Jong-Min
    • 한국발생생물학회지:발생과생식
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    • 제24권3호
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    • pp.231-239
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    • 2020
  • Many benefits of silk protein fibroin (SPF) have been suggested in biomedical applications; and notably, significant SPF effects have been observed for metabolic syndromes that are directly linked to insulin resistance, such as type 2 diabetes mellitus (T2DM). Based on our previous findings, we believe that SPF from spiders exhibits outstanding glucose-lowering effects in diabetic BKS.Cg-m+/+Leprdb mice. In order to evaluate the dietary effects of SPF in diabetic animals, we generated several lines of transgenic rice (TR) that expresses SPF, and the feeding of TR-SPF to diabetic animals decreased blood glucose levels, but did not change insulin levels. Western blot analyses of hepatic proteins showed that AMP-activated protein kinase (AMPK) expression and phosphorylation both decreased in TR-SPF-fed groups, compared with controls. This finding suggests that the glucose-lowering effects in this diabetic animal model might be AMPK-independent. In contrast, six-transmembrane protein of prostate 2 (STAMP2) was upregulated after TR-SPF exposure. Together with STAMP2, the Akt protein phosphorylation increased after TR-SPF exposure, which indicates that STAMP2 leads to Akt phosphorylation and thus increases insulin sensitivity in hepatocytes. Importantly, the hepatic steatosis that was seen in the liver of diabetic mice was remarkably alleviated in TR-SPF-fed mice. Hepatocytes that were immunopositive for STAMP2 were overwhelmingly observed in hepatic tissues from TR-SPF-fed mice compared to the control. Taken together, these results suggest that feeding diabetic mice with TR-SPF upregulates STAMP2 expression and increases Akt phosphorylation in hepatic tissues and thus potentially alleviates insulin resistance and hepatic steatosis.