A rapid and sensitive method for measuring monooxygenase activities in hepatocytes cultured in 96-well plates

Summary Measurement of biotransformation activities in cells is of great importance for drug metabolism and toxicologic studies. It is currently done by measuring the enzymatic activities in partially purified microsomes. In the present work we report on a rapid, easy, sensitive, and reproducible fluorimetric assay for quantifying cytochrome P450-dependent monooxygenase activities (P450IA1, P450IIB1) in hepatocytes cultured in 96-well plates. The procedure involves the direct determination of enzymatic activities in intact hepatocytes while avoiding cell homogenization, thereby permitting use of a the reduced number of cells and allowing cultured cells to be used in later experiments. Substrates (7-ethoxyresorufin, 7-pentoxyresorufin) are added to culture medium and metabolized by hepatocytes. After enzymatic deconjugation, the fluorescent resorufin present in culture medium is quantified by means of a microplate fluorimetric reader. Major advantages of this technique, as compared to other available methods, are: a) no cell disruption is required; b) activity can be measured with a very small number of cells; c) rapid processing time; and d) possibility of performing repeated assays with the same cell monolayer.     

彩色多普勒在心脏起搏器综合征诊断中的应用价值

目的 :探讨运用彩色多普勒血流显像 (CDFI)并结合心电图 (ECG)诊断起搏器综合征 (PMS)的临床价值。方法 :对具有详实临床及ECG资料的心室按需 (VVI)起搏器安置患者 6 0例及对照组 4 8例进行CDEI检查 ,观察起搏组与对照组的三尖瓣返流 (TR)的发生率、返流程度 ,并分析二者之间的相关性。结果 :起搏组出现逆传P波 (即PMS阳性 )者 15例 ,CDFI检测出现TR者 2 0例 ,明显高于对照组 ,且PMS阳性者TR发生率明显高于PMS阴性者 ,表明安置VVI起搏器后出现逆传P波与TR的发生相关性良好。结论 :CDFI对PMS的诊断具有重要意义 ,尤其将ECG与CDFI结合应用有助于诊断率的提高。     

Interaction between thiol-modifying agents and P1075, a KATP channel opener, in rat isolated aorta

In vascular smooth muscle, openers of ATP-dependent potassium channels (K _ATP channels), such as P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the ⁸⁶Rb⁺ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in ⁸⁶Rb⁺ efflux induced by P1075 was taken as a qualitative measure of K⁺ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100μM), abolished specific binding of [³H]-P1075 with an IC₅₀ value of 7.6±1.2μM; at 30μM, the half time for inhibition was 38min. Two other thiol-oxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2’-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1mM), reduced [³H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In ⁸⁶Rb⁺ efflux experiments, thimerosal (3 to 100μM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC₅₀ value of 7±1μM. The inhibitory effect occurred with a half-time of approximately 8min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation.The data show that oxidation of thiol groups interferes with the binding of the K _ATP channel opener, P1075; concomitantly, the ⁸⁶Rb⁺ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K_ATP channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated ⁸⁶Rb⁺ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity. Received: 7 April / Accepted: 9 July 1997     

5-羟色胺对大鼠脊髓P物质痛觉调制的影响(英文)

比较SP,5-HT与For诱发的脊髓内c-fos表达的异同,以及它们之间的相互关系,从而进一步了解SP在脊髓痛觉调制中的主要作用.方法:用免疫组织化学法和痛阈测定法.结果:发现大鼠it P物质(sP)10μg和sc5％甲醛(For)150μL诱发的脊髓c-fos表达主要在背角Ⅰ,Ⅱ,Ⅴ及Ⅵ层,同时SP使痛阈降低,For使痛级均数(PIR)升高.5-HT it 20μg引起的c-fos表达较多地分布于背角Ⅲ—Ⅳ层,并可使痛阈升高.5-HT和Fen可分别减弱和增加SP及For诱发的脊髓c-fos表达及痛反应.结论:SP在脊髓内可能主要起致痛作用,5-HT可抑制SP引起的脊髓c-fos表达,从而参与SP的痛觉调制作用.     

Evidence for the presence of substance P-like immunoreactivity in the human cerebellum

Preliminary results on the localization of substance P-like immunoreactivity in the human cerebellum are presented. Cerebella from newborn and adult subjects were examined. While only sporadic positive fibres were detected in the adult tissue, the immunoreactive material appeared more abundant in the cerebella from newborn subjects. Varicose and non-varicose fibres and dot-like nerve terminals were present with different density in various regions. The paucity of immunoreactive perikarya suggests that most of the cerebellar substance P-like immunoreactive material has an extrinsic origin.     

Nd：YAG激光对牙本质钙／磷比值的影响

为探讨Ｎｄ：ＹＡＧ激光对牙本质矿物含量的影响，应用扫描电镜和能谱仪定点测量５０个上前牙标本的根管壁（激光照射工和正常对比区）的钙、磷含量，结果表明，照射区牙本质钙／磷（Ｃａ／Ｐ）比值高于对比区，当激光能量达１５Ｗ以上时变化更为显著，提示Ｎｄ：ＹＡＧ激光能提高牙本质的钙化程度。     

RP—HPLC用于紫苏油及其习用品白苏油,野苏油的鉴别

本文采用RP－HPLC方法对紫苏油及其习用品白苏油、野苏油甘油酯进行了定性定量分析。利用HPLC分析油脂也为中药材真伪鉴别提供了一条有效途径。     

乳腺癌中P73、P21的表达及其意义

目的 :通过检测 P73蛋白、P2 1蛋白在乳腺癌中的表达及其与临床病理指标的关系 ,探讨乳腺癌的生物学特性 ,分析它们在乳腺癌的发生、转移中所起的作用。方法 :应用免疫组化 S-P法检测 68例乳腺癌石蜡包埋标本中 P73、P2 1的表达情况。结果 :a.在乳腺癌 - 级与 级之间 P73蛋白阳性率分别为 1 8.75 % ( 9/48)和 60 .0 0 % ( 1 2 /2 0 ) ,差异有显著性 ( P <0 .0 5 )。随着 P73阳性表达率增高 ,乳腺癌的恶性程度增高。b.淋巴结转移组 p2 1的阳性表达率 2 3 .3 3 % ( 7/3 0 )明显低于无淋巴结转移组的阳性率 5 2 .63 % ( 2 0 /3 8) ,差异有显著性( P <0 .0 5 )。c.P73、P2 1之间无相关。结论 :P73、P2 1与乳腺癌的发生、发展有关     

正规康复治疗对脑卒中患者P300的影响

目的研究正规康复治疗对脑卒中患者P300的影响,为脑卒中患者认知功能的康复提供临床依据.方法按照统一的入选条件选取2001年10月～2002年2月在本院住院的脑卒中患者60例为研究对象,随机分成康复组和对照组,康复组在脑卒中后3～7 d开始康复治疗,为期1个月.对照组未给予康复治疗,治疗前后进行P300检查.结果康复组在康复后与对照组及在康复后与康复前相比,P300的潜伏期明显缩短,波幅明显增高(P＜0.01).结论正规康复治疗可以明显改善脑卒中患者的认知功能.