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.     

Learners’ acceptance of a webinar for continuing medical education

The aim of this study was to evaluate learners’ acceptance of a webinar for continuing medical education that was instigated by the International Association of Oral and Maxillofacial Surgeons (IAOMS). A live, interactive webinar on orthognathic surgery was broadcast via the Internet. The learners’ acceptance of the webinar was evaluated using a standardized, validated questionnaire (Student Evaluation of Educational Quality, SEEQ). One hundred and fifty-three participants attended the webinar; 55 participants (46 male, nine female) completed the questionnaire. The mean age of the respondents was 41.6 ± 10.0 years. The age of male and female respondents did not differ significantly. The respondents were spread over five continents, with the highest number from Brazil. The SEEQ showed a high level of acceptance for almost all subscales. There was no statistically significant difference between male and female respondents concerning acceptance of the webinar (P = 0.614). The wide distribution of participants shows the potential for webinars as facilitators of barrier-free distribution of knowledge. The webinar was well accepted by the attendees independent of sex, specialty, and work experience. However, the sex ratio reflects the underrepresentation of women in oral and maxillofacial surgery.     

高转换型肾性骨病中骨保护素及其配体的表达和形态计量学观察

目的 观察高转换型肾性骨病中骨保护素及其配体 (OPG，RANKL)的表达，并与骨形态计量学指标进行相关分析。 方法 选择10例慢性肾衰尿毒症患者和3例正常人进行髂骨活检术，获得骨组织标本。采用免疫组化方法检测OPG和RANKL蛋白质的表达。采用全自动图像分析系统进行骨组织形态计量学测定。结果 10例慢性肾衰尿毒症患者经骨病理学检查证实均为高转换型骨病，以破骨细胞活化形成骨吸收陷窝伴或不伴骨矿化不全为特点。免疫组化显示尿毒症患者骨组织中以RANKL阳性表达为主。与正常对照相比，RANKL阳性表达细胞数目显著增加，OPG阳性表达细胞数目显著减少。尿毒症患者RANKL的阳性表达细胞数目与骨吸收面积和破骨细胞数目呈显著正相关。结论 高转换型肾性骨病中，PTH的溶骨作用可能是通过OPG/RANKL/RANK系统介导的。     

白细胞介素-18基因修饰的胎肝细胞经脾移植对小鼠免疫功能的影响

目的：观察表达mIL-18的重组腺病毒基因修饰的胎肝细胞（AdmIL-18/BNL.CL2)经脾移植对正常小鼠免疫功能的影响。方法：实验组小鼠经脾移植AdmIL-18/BNL.CL2，同时设LacZ病毒对照组（Ad-LacZ/BNL.CL2),BNL.CL2细胞对照组及空白对照组。2周后处死，留取血清，制备腹腔巨噬细胞、脾淋巴细胞、肝组织匀浆液，提取肝组织总RNA。采用ELISA法检测各组小鼠血清、腹腔Mφ和脾细胞培养上清、肝匀浆中细胞因子的含量；采用半定量RT－PCR法，检测肝组织细胞因子mRNA相对表达量；以LDH释放法测定腹腔Mφ杀伤活性和脾NK细胞活性，用MTT还原比色法测定脾淋巴细胞的增殖活性。结果：实验组小鼠血清、细胞培养上清及肝匀浆中，IL－18、IL－2、IFN－γ、TNF-α稔均高于其它对照组，而IL－4、IL－10水平则低于对照组；半定量RT－PCR结果与ELISA检测结果一致；同时，实验组腹腔Mφ的杀伤活性和脾NK细胞活性，及脾淋巴细胞增殖活性也明显高于对照组。结论：AdmIL-18能有效转染至胎肝细胞并稳定表达mIL-18;AdmIL-18基因修饰的胎肝细胞经脾移植后，可显著提高肝脏、脾脏免疫细胞活性，活化腹腔Mφ，促进Th1类细胞因子表达，抑制Th2类细胞因子的分泌。     

用逆转录—聚合酶链反应法对类胰岛素生长因子cDNA的扩增

逆转录-聚合酶链反应(RT-PCR)法是将PCR方法与逆转录法结合应用,可快速高效地扩增某一基因的全cDNA。类胰岛素生长因子-I(IGF-I)在人染色体中只有一个基因,全长超过45kb,并有内含子,其cDNA却只有几百个bp,为了表达得到IGF-I,本文利用RT-PCR方法,从胎肝提出的混合mRNA中,筛选并扩增了IGF-I的cDNA。经过分子克隆与测序,证明不需制备胎肝cDNA库,即可快速获得IGF-I的cDNA。     

18F-FDG符合线路显像与同机CT融合在肿瘤诊断的应用

【目的】评价~(18)F-FDG符合线路显像及同机CT图像融合在肿瘤诊断中的价值。【方法】经病理确诊或临床确诊为恶性肿瘤的病人79例,良性肿块病人19例进行~(18)F-FDG符合线路显像及同机CT图像融合检查,经COSEM重建,结果与解剖显像比较。【结果】①~(18)F-FDG符合显像诊断恶性肿瘤的敏感性、特异性、准确率、阳性预测值和阴性预测值分别为92.4％、89.5％、91.8％、97.3％和73.9％,解剖显像分别为79.8％、63.2％、76.5％、90.0％和42.9％。②同部位显像比较中,~(18)F-FDG显像病灶探测数为302个,而解剖显像为286个。③同机CT融合明显提高对肺部近纵隔、膈缘及腹部等部位的定位诊断。【结论】~(18)F-FDG符合线路显像较解剖显像更好地对恶性肿瘤作出鉴别诊断,并且能探测到更多的恶性病灶,同机CT融合较大地提高定位诊断的准确性,具有重要的临床价值,值得临床广泛应用。     

18氟-氟化脱氧葡萄糖在下咽部肿瘤诊断中的作用

目的：研究^18氟—氟化脱氧葡萄糖(^18F-FDG)在下咽部肿瘤诊断中的作用。方法：20例下咽部肿瘤患者均接受^18F-FDG检查及CT、全组内镜(食管、咽喉、气管镜)检查。结果：20例下咽部肿瘤患者^18F-FDG检查结果显示：2例阴性；18例肿瘤原发部位有同位素浓集，其中5例伴异位同位素浓集。对此5例患者进一步临床、病理检查证实：1例食管部占位，2例肺部病变，2例为良性改变。^18F-FDG检查结果显示的病变范围与CT、内镜检查相符。结论：^18F-FDG检查可以提高下咽部肿瘤、尤其是隐匿的并发肿瘤诊断的准确性，明确病变范围，为临床治疗提供依据；同时又是一个简单、易行的下咽部肿瘤的随访检查手段。     

17β-雌二醇对子宫内膜异位症患者在位子宫内膜间质细胞β-catenin mRNA和蛋白表达的影响

目的研究17β-雌二醇(17β-E2)对子宫内膜异位症(内异症)患者在位子宫内膜间质细胞β-catenin mRNA和蛋白表达的影响,探讨Wnt/β-catenin信号通路在介导雌激素促进内异症发生发展的作用。方法体外分离培养内异症患者在位子宫内膜间质细胞。用不同浓度17β-E2处理子宫内膜间质细胞48 h;此后选用10-10mol/L 17β-E2处理子宫内膜间质细胞12、24和48 h,逆转录聚合酶链反应(RT-PCR)和免疫印迹法(Western blotting)检测17β-E2处理前后子宫内膜间质细胞β-catenin mRNA和蛋白的表达水平。同法分析雌激素受体拮抗剂ICI182,780(10-6mol/L)对17β-E2促进β-catenin mRNA和蛋白表达的影响。免疫组织化学染色观察17β-E2作用后β-catenin在子宫内膜间质细胞中的定位。结果17β-E2能明显促进内异症患者在位子宫内膜间质细胞β-catenin mRNA和蛋白的表达,并呈剂量和时间依赖性,于10-10mol/L作用48 h最明显。雌激素受体拮抗剂ICI182,780能明显抑制17β-E2对子宫内膜间质细胞β-catenin mRNA和蛋白的表达。免疫组织化学染色发现17β-E2能促进β-catenin在子宫内膜间质细胞核内的表达。结论雌激素可能通过激活Wnt/β-catenin信号通路促进内异症在位子宫内膜的异位种植。     

LHRH-PE40识别结肠癌细胞膜表面蛋白的研究

目的探讨人结肠癌细胞系Lovo及人白血病细胞系Jurket细胞膜表面蛋白能否识别LHRH-PE40及是否存在竞争性抑制。方法将结肠癌细胞系Lovo及白血病细胞系Jurket制备成细胞膜,利用125I标记的LHRH-PE40与两种细胞膜进行放射性配基分析,与LHRH进行竞争结合分析。结果人结肠癌细胞系Lovo的结合竞争符合特异性配基-受体结合、竞争;而白血病细胞系Jurket未见配基-受体特异性结合。其中LHRH-PE40与Lovo细胞的亲和力:Kd=10·6±2·33nmol/L,容量Bmax=345±7·59pmol/mg。结论LHRH是结肠癌免疫治疗的有效靶点,LHRH-PE40对过度表达LHRH受体的结肠癌具有特异性杀伤作用,而对无LHRH表达的肿瘤无杀伤作用,对于药物的临床应用有着指导意义。