Carboxylesterase catalyzed 18O-labeling of carboxylic acid and its potential application in LC-MS/MS based quantification of drug metabolites

LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel ¹⁸O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H₂¹⁸O in the presence of the enzyme, generating singly- and doubly-¹⁸O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds – indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery.     

谷胱苷肽S转移酶在口腔鳞癌中的表达

目的　研究异型增生程度不同的口腔白斑和不同分级的鳞癌中谷胱苷肽S转移酶π(GST π)的表达 ,探讨GST π在口腔鳞癌发生发展中的作用。　方法　采用免疫S -P法 ,对 5 4例轻、中、重度异型增生 ,4 7例高、中、低分化的口腔鳞癌 ,7例口腔粘膜上皮单纯增生患者组织进行GST π检测。　结果　口腔粘膜上皮单纯增生组织中未见GST π的表达 ,轻、中、重度异型增生病例中的GST π阳性率分别为4 7.8%、5 2 .9%和 6 4 .2 % ,高于单纯增生组 (P <0 .0 1) ;高分化鳞癌GST π阳性率为 6 4 .7% ,中、低分化鳞癌组分别为 2 8.5 %及 2 2 .2 % ,中、低分化鳞癌组表达均低于高分化鳞癌组及异型增生组 (P <0 .0 5 )。　结论　GST π表达的变化与口腔鳞癌早期的发生发展密切相关     

The participation of adenosine receptors in the adenosine 5''-triphosphate-induced relaxation in the isolated rabbit corpus cavernosum penis

AIM: To investigate the participation of adenosine receptors in the adenosine 5'-triphosphate (ATP)-induced relaxation in the corpus cavernosum penis (CCP) of rabbits. METHODS: The ATP-induced relaxation was assessed on the noradrenaline precontracted CCP of rabbits in the presence and absence of 8-(3-chlorostyryl)caffeine (CSC); an adenosine A(2A) receptor antagonist; alloxazine and MRS1754; adenosine A(2B) receptor antagonists; and ARL67156, an inhibitor of ecto-nucleoside triphosphate diphosphohydrolases. RESULTS: Adenosine and ATP relaxed the noradrenaline precontracted CCP of rabbits in a concentration-dependent manner. The adenosine- and ATP-induced relaxations were suppressed by alloxazine and MRS1754, but not by 8-(3-chlorostyryl)caffeine. ARL67156 potentiated the ATP-induced relaxation but not the adenosine-induced one. MRS1754 suppressed the ATP-induced relaxation potentiated by ARL67156. CONCLUSIONS: The above results suggest that, in the CCP of rabbits, the adenosine receptor mediating adenosine-induced relaxation is of the A(2B) receptor and the ATP directly causes relaxation through the A(2B) receptor on the CCP.     

Alteration of Human Placental Vascular Tone by Antiarrhythmic Medications In Vitro

Antiarrhythmic and Placental Vessels. Introduction : Antiarrhythmic medications are commonly used during pregnancy for treatment of maternal or fetal arrhythmias, but little is known about their effect on human placental vascular tone and, consequently, placental blood flow. The objective of this study was to evaluate the tone responses caused by antiarrhythmic medications in human placental vessels from normal term pregnancies in vitro.
Methods and Results : Isolated human placental arteries and veins from uncomplicated term pregnancies incubated in Krebs'-bicarbonate under 5% oxygen/5% carbon dioxide/balance nitrogen (PO₂ 35 to 38 torr) were exposed to cumulative doses of quinidine, procainamide, lidocaine, flecainide, propranolol, amiodarone, verapamil, digoxin, and adenosine after submaximal contraction with 5-hydroxytryptamine. The study was conducted both in the presence and absence of endothelium. The addition of the tested medications caused a significant, dose-dependent relaxation of human placental arteries and veins except for adenosine, which induced a sustained, dose-dependent contraction of human placental vessels regardless of the presence or absence of tone. Removal of the endothelium did not alter these responses.
Conclusions : Based on these results, the medications tested should have no decremental effect on placental blood flow, with the possible exception of adenosine, which causes significant. dosedependent contraction of human placental vessels in vitro. Should similar contraction be present in vivo, it may have an adverse effect on the fetus when administering adenosine to pregnant women at term or during labor.     

三磷酸腺苷-氯化镁冷稀释血停搏液的实验研究及临床应用

用液体闪烁计数法测定离体再灌注兔心肌线粒体内４５Ｃａ２＋的放射性强度．观察三磷酸腺苷－氯化镁冷稀释血停搏液和冷稀释血停搏液对缺血再灌注兔心肌线粒体内４５Ｃａ２＋的影响．结果表明，在３０ｍｉｎ以内使用冷稀释血停搏液组兔心肌线粒体内４５Ｃａ２＋放射性强度高于三磷酸腺苷－氯化镁冷稀释血停搏液组（Ｐ＜０．０５），而６０ｍｉｎ时冷稀释血停搏液组的放射性强度稍高或接近于三磷酸腺苷－氯化镁冷稀释血停搏液组，差异无显著性（Ｐ＞０．０５）．在２７例心内直视手术中使用三磷酸腺苷－氯化镁冷稀释血停搏液均获良好效果．     

INTERACTION BETWEEN EPIDERMAL GROWTH FACTOR AND ARGININE VASOPRESSIN IN RENAL MEDULLARY MEMBRANES

1. Epidermal growth factor is a potent mitogen that causes natriuresis, diuresis and inhibition of arginine vasopressin-induced water reabsorption. 2. The aim of this study was to determine any interaction between epidermal growth factor and the V1 (vascular) and/or V2 (antidiuretic) arginine vasopressin receptor subtypes. 3. Radioligand binding displacement assays demonstrated that although arginine vasopressin related peptides displaced both radioligands from renal medullary membranes at low concentrations epidermal growth factor displaced neither. 4. Arginine vasopressin V2 receptor second messenger cyclic adenosine monophosphate (CAMP) production was inhibited by epidermal growth factor (IC₅₀ 2 ± 10^?7 mol/L) as was sodium fluoride cAMP production but only at much higher concentrations. 5. Therefore the diuretic effect of epidermal growth factor is not via direct antagonism of arginine vasopressin receptors but seems mediated via inhibition of the V2 second messenger system.     

胎盘型谷胱甘肽S转移酶mRNA在大鼠肝癌癌前病变组织中的原位表达

应用原位杂交技术，观察了二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌前病变组织中胎盘型谷胱甘肽Ｓ转移酶（ＧＳＴ－Ｐ）ｍＲＮＡ的表达。结果显示，ＧＳＴ－ＰｍＲＮＡ主要在癌前病变肝组织中的变异灶及灶外卵圆形细胞内表达，且在变异灶间或和同一灶内阳性细胞间表达程度不尽一致，而正常肝、再生肝组织中未见其表达。提示在分子水平上变异灶细胞及卵圆型细胞可能成为实验性肝癌的癌前期细胞     

重组O-GLcNAc糖基转移酶在昆虫细胞中的表达和纯化

蛋白质的 O- Glc NAc糖基化修饰是一种细胞核蛋白与细胞浆蛋白的蛋白质翻译后修饰。不同于膜蛋白和分泌蛋白的糖基化修饰 ,与蛋白质磷酸化修饰相似。催化蛋白质 O- Glc NAc糖基转移酶 ( OGT)已被克隆。用Hi5昆虫细胞系统表达并纯化了具有活性的重组 OGT。在 Hi5昆虫细胞中表达的鼠肝 OGT带有一 His 6标记片段。经镍离子螯合柱纯化后 ,其比活性达到 0 .88nmol· min- 1·m g- 1 OGT。因此本研究为进一步研究蛋白 O- Glc NAc糖基化修饰提供重要资源