• Biomolecules & Therapeutics
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• 제2권4호
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• pp.361-368
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• 1994

The nature of the muscarinic receptors in bovine adrenal medulla was investigated in this study. [$^3$H]Quinuclidinyl benzilate(QNB) specifically bound to a single class of muscarinic receptor with a $K_{D}$ value of about 70 pM in bovine adrenal medullary, cardiac ventricular and ileal homogenates. Pirenzepine inhibition curves of [$^3$H]QNB binding to cardiac ventricular and ileal homogenates were steep, indicating the presence of a single class of binding site for pirenzepine with a Ki value of 990 nM and 508 nM, respectively. However, pirenzepine/[$^3$H]QNB competition binding curves in adrenal medulla suggested the presence of two binding sites (Hill coefficient=0.59) with a high( $M_1$) and a low( $M_2$) affinity. Respective Ki values for pirenfepine were 16 nM and 633 nM, with 44% of total sites having a high affinity( $M_1$). Gallamine, which is selective to cardiac $M_2$-receptor, inhibited [$^3$H]QNB binding to adrenal medullary, cardiac ventricular and ileal homogenates with Ki values of 12 $\mu$M, 6 $\mu$M and 13 $\mu$M, respectively. Thus, the binding affinities of pirenzepine and gallamine for $M_2$-receptor in adrenal medulla were similar to those in ileum, which contains the $M_3$-receptor. These results indicate that the $M_1$- and $M_3$- muscarinic receptor subtypes coexist in the bovine adrenal medulla.a.

• Asian Pacific Journal of Cancer Prevention
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• 제13권3호
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• pp.937-940
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• 2012

Due to lack of sufficient data on characteristics of breast cancer patients and risk factors for developing metastasis in Iran this study was designed to understand clinical aspects impacting on survival. A cross-sectional study on breast cancer patients was conducted in an oncology clinic of the university hospital between 1995 and 2010. Data were retrieved from medical records and included age, menopausal status, tumor diameter, number of involved nodes, histopathological type, estrogen and progesterone receptor expression, c-erbB-2, primary and secondary metastasis sites, overall survival, disease free interval and type of chemotherapy protocol. The results were analyzed with SPSS 13 software. The mean age of the patients was 49.2 (27-89) years. The primary tumors were mainly ER positive (48%) and PR negative (49.3%). The status of lymph nodes dissected and examined in these patients was unknown in 19 patients (25.3%) while 18 patients (24%) had positive lymph nodes with no report on the number of involved nodes. All of the patients had received antracyclin based chemotherapy in an adjuvant or metastatic setting. Adjuvant hormonal therapy was administered to receptor positive patients. In average, overall survival after recurrence was 30 months (95%CI 24.605-35.325) for non-skeletal versus 42 months (95%CI 31.211-52.789) for skeletal metastasis (P= 0.002). The median survival was also greater for receptor positive patients; 39 months (95%CI 33.716-44.284) for PR+ versus 26 months (95%CI 19.210-32.790) for PR- (P=0.047) and 38 months (95%CI 32.908-43.092) for ER+ versus 27 months (95%CI 18.780-35.220) for ER- patients (P=0.016). No relation was found between site of first metastasis and hormone receptor, age, tumor diameter, DFI and menopausal status. Sites of metastasis were independent of age, size of the tumor, menopausal and hormone receptor status in this study. Overall survival provided significant relations with respect to receptor status and bone metastasis.

• 약학회지
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• 제34권4호
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• pp.224-237
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• 1990

A single uniform population of specific, saturable, high affinity binding site of $[^3H]QNB$ guinuclidinyl benzilate(QNB) was identified in the rat cerebral microsomes. The Kd value(37.2 pM) for $[^3H]QNB$ calculated from the kinetically derived rate constants was in agreement with the Kd value(48.9 pM) determined by analysis of saturation isotherms at various receptor concentrations. Dimenhydrinate(DMH), histamine $H_1-blocker$, increased Kd value for $[^3H]QNB$ QNB without affecting the binding site concentrations and this effect resulted from the ability of DMH to slow $[^3H]QNB-receptor$ association. Pirenzepine inhibition curve of $[^3H]QNB$ binding was shallow(nH = 0.52) indicating the presence of two receptor subtypes with high ($M_1-site$) and low($M_2-site$) affinity for pirenzepine. Analysis of these inhibition curves yielded that 68% of the total receptor populations were of the $M_1-subtype$ and the remaining 32% of the $M_2-subtype$. Ki values for the $M_1-$ and $M_2-subtypes$ were 2.42 nM and 629.3 nM, respectively. Ki values for $H_1-blockers$ that inhibited $[^3H]QNB$ binding varied with a wide range ($0.02-2.5\;{\mu}M$). The Pseudo-Hill coefficients for inhibition of $[^3H]QNB$ binding by most of $H_1-blockers$ examined except for oxomemazine inhibition of $[^3H]QNB$ binding were close to one. The inhibition curve for oxomemazine in competition with $[^3H]QNB$ was shallow(nH = 0.74) indicating the presence of two receptor populations with different affinities for this drug. The proportion of high and low affinity was 33:67. The Ki values for oxomemazine were $0.045{\pm}0.016\;{\mu}M$ for high affinity and $1.145{\pm}0.232\;{\mu}M$ for low affinity sites. These data indicate that muscarinic receptor blocking potency of $H_1-blockers$ varies widely between different drugs and that most of $H_1-blockers$ examined are nonselective antagonist for the muscarinic receptor subtypes, whereas oxomemazine might be capable of distinguishing between subclasses of muscarinic receptor.

• 미생물학회지
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• 제47권1호
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• pp.7-13
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• 2011

일반적으로 대장균을 숙주로 이용하여 고등생물 유래 막단백질을 발현시킬 경우 발현된 막단백질은 숙주 세포에 치명적인 독성을 보인다. 우리가 발현을 시도한 15개의 인간 막단백질 중에서 특히 퓨린수용체 $P2X_4$ 발현은 대장균에 강한 독성을 보였다. 이러한 독성의 원인을 알아보기 위해 hydroxylamine을 사용하여 하여 인간 $P2X_4$ 유전자를 돌연변이 시키고 독성이 약해진 돌연변이체를 선별하였다. 돌연변이체 단백질을 면역블랏으로 분석한 결과 야생형에 비해 모두 단백질의 크기가 작았다. 크기가 제법 큰 돌연변이 두 개를 골라 DNA 서열분석을 해보니 130번째, 또는 194번째 Trp 코돈이 종결코돈으로 바뀜으로써 두 번째 막통과 도메인이 사라진 truncated protein이라는 사실을 알았다. 이들 돌연변이체의 세포내 위치를 추적해보니 둘 다 세포막에 삽입되어 있지는 않았다. 이런 결과를 종합해 볼 때 $P2X_4$의 발현이 대장균에 독성을 보이기 위해서는 전체 단백질의 올바른 세포막 삽입이 중요함을 시사한다.

• 한국발생생물학회지:발생과생식
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• 제11권3호
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• pp.187-193
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• 2007

Ethane 1,2-dimethane sulfonate(EDS)는 Leydig 세포의 선별적 사멸을 유도하는 약물로서 가역적인 테스토스테론 결핍 흰쥐를 만드는데 널리 사용된다. 부정소의 구조와 기능 유지는 크게 보아 정소에서 분비되는 테스토스테론에 의존적이지만, 테스토스테론으로부터 유도되는 dihydroxytestosterone(DHT)와 에스트로겐도 중요한 역할을 한다. 본 연구에서는 EDS 주사 후 7주까지 부정소에서의 스테로이드 호르몬 수용체, cyctochrome P450aromatase(P450arom)와 $5{\alpha}$-reductase의 유전자 발현 양상을 조사하였다. 성숙한 수컷 흰쥐($350{\sim}400\;g$)에 EDS를 1회 복강 주사하고(75 mg/kg i.p.) 주사 후 0, 1, 2, 3, 4, 5, 6, 7주가 경과한 날에 희생하였다. 표적 유전자들의 전사 활성은 반 정량적 역전사 중합효소 반응법(semi-quantitative RT-PCRs)으로 측정하였다. Estrogen receptor alpha($ER{\alpha}$) 전사 수준은 EDS 실험군에서 대조군에 비해 주사 1주후에 유의하게 상승했으나(P<0.01) 2주 후부터는 대조군과 유의적인 차이를 보이지 않았다. Estrogen receptor beta($ER{\beta}$)의 전사 수준은 주사 1주후 EDS 실험군에서 대조군에 비해 유의하게 증가했다가(P<0.05), 2주와 3주에는 감소하였고(P<0.05와 P<0.01), 4주와 6주까지는 변동폭을 보이다가 7주 후에는 대조군에 비해 증가하였다(P<0.05). Androgen receptor(AR) 전사 수준은 주사 2주 후에 유의하게 증가하다가(P<0.01) 3주 후부터는 대조군 수준으로 회복하였다. 반면, P450arom는 주사 1주 후부터 3주까지 급격하게 감소했다가(P<0.01 1주와 2주; P<0.05 3주), 4주에 대조군 수준으로 회복하였다. $5{\alpha}$-reductase type 2($5{\alpha}$-RT2)의 mRNA 수준은 4주 후 유의하게 증가했다가(P<0.01), 이후 대조군 수준으로 회복하였다. 본 연구는 EDS 주사가 성 스테로이드 호르몬 수용체들과 안드로겐 전환 효소들의 전사 활성에 가역적인 변화를 유도함을 보여준 것이다. EDS 주사 모델은 부정소의 생리 조절 기작을 이해하는데 유용할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.247-254
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• 2015

In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

• KSBB Journal
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• 제28권1호
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• pp.7-12
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• 2013

Thromboxane $A_2$ ($TXA_2$) is one of major proinflammatory mediators, plays an important role in the development of vascular inflammatory diseases. $TXA_2$ acting through the thromboxane receptor regulates multiple pathways and genes in a variety of cells. In this study, we report that the activation of thromboxane receptor with U46619 increases the interleukin-8 (IL-8) mRNA in vascular endothelial cells. We also demonstrated that U46619 produces the activations of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK), which is required for endothelial IL-8 production. And U46619 enhanced mRNA stability of IL-8 transcripts in endothelial cells. Moreover, inhibition of ERK1/2 or p38MAPK reduced monocyte adhesion to aortic endothelium stimulated by U46619. Therefore, these results suggest that activation of thromboxane receptor promotes the expression of IL-8 via ERK1/2 and p38MAPK activation in endothelial cells.

• 약학회지
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• 제32권2호
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• pp.101-111
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• 1988

$[^3H]$ Quinuclidinyl benzilate(QNB) binding assays were performed in the dog ventricular sarcolemma fraction enriched approx. 32-fold in sarcolemma compared to the starting homogenate to elucidate the effect of antihistaminics on cardiac muscarinic receptor. Chlorpheniramine(CHP) inhibited specific binding of $[^3H]$QNB and delayed the equilibrium binding. The rate constants at $37^{\circ}C$ for formation and dissociation of the QNB receptor complex were $0.38{\times}10^9\;M^{-1}$ and $1.6{\times}10^{-2}\;min^{-1}$, respectively. The mean value for the dissociation constant from the pairs of the rate constants was 43. 2 pM and this value was similar to the value(44.8pM) determined from Scatchard analysis. CHP decreased association rate constant, indicating increase in $K_D$ value. Decrease in affinity without affecting the binding site concentration$(B_{max})$ for $[^3H]$QNB binding by CHP was also demonstrated by Scatchard analysis. $K_i$ values for $H_i$-blockers that inhibited specific $[^3H]$QNB binding were $0.02{\sim}4.8{\mu}M$. Cimetidine with $K_i$ value of $230{\mu}M$, however, was ineffective in displacing $[^3H]$QNB binding at concentration of $50{\mu}M$. The Hill coefficient for $H_1$-blockers were about one. The results indicate that $H_1$-antihistaminics inhibit $[^3H]$ QNB binding by interaction with myocardiac muscarinic cholinergic receptor and anticholinergic side effects of these drugs are mainly due to this receptor blocking mechanism.

• Tuberculosis and Respiratory Diseases
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• 제74권6호
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• pp.264-268
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• 2013

Background: Idiopathic pulmonary fibrosis (IPF) is a lethal pulmonary fibrotic disease. In general, the exaggerated activation of the coagulation cascade has been observed during initiation or maintenance of the fibrotic disease. In our recent study, immunohistochemical expression of protease-activated receptor-2 (PAR-2), which plays a key role in coagulation cascade, was observed in surgical specimen of IPF patients, and associated with poor clinical outcome. The aim of this study was to evaluate the overexpression of PAR-2 in inflammatory cells from peripheral blood and bronchoalveolar lavage fluid in IPF patients. Methods: From May 2011 to March 2012, IPF patients and controls were enrolled in Seoul National University Hospital. Peripheral blood and bronchoalveolar lavage fluid were collected for analysis of PAR-2 expression. Flow cytometry and reverse transcription polymerase chain reaction were used for PAR-2 receptor and mRNA assessment. Results: Twelve IPF patients and 14 controls were included in this study. Among them, flow cytometry analysis was conducted from 26 peripheral blood (patient group, 11; control group, 13) and 7 bronchoalveolar lavage fluid (patient group, 5; control group, 2). The expression of PAR-2 receptor was not different between patient and control groups (p=0.074). Among all 24 population, PAR-2 mRNA assessment was performed in 19 persons (patient group, 10; control group, 9). The mRNA expression of PAR-2 was not significant different (p=0.633). Conclusion: In IPF patients, PAR-2 receptor and mRNA expression were not different from control group.

• Animal cells and systems
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• 제7권2호
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• pp.145-149
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• 2003

Adult periodontitis is a chronic inflammatory disease whose etiology is not well defined. Recent studies have shown that vitamin D receptor gene has been a candidate for the susceptibility of adult periodontitis. The purpose of this study is to investigate the frequency of Taq I restriction fragment length polymorphism (RFLP) in the vitamin D receptor gene in nan periodontically healthy controls and 28 adult periodontitis patients. Taq I RFLP in the vitamin D receptor gene was detected by PCR amplification, followed by restriction enzyme digestion and 2％ agarose gel electrophoresis. There was no significant difference in the distribution of Taq I RFLP between healthy controls and adult periodontitis group (P > 0.05). Thus, Taq I RFLP in the vitamin D receptor gene may not confer the susceptibility to adult periodontitis in Korean population. However, t allele distributions of this RFLP showed various frequencies among ethnic groups studied. Further studies in other ethnic groups will be required.