延髓—脊髓P物质能神经元下行通路参与毒扁豆碱的中枢升压反应

大鼠延髓腹侧面头端应用毒扁豆碱引起血压升高和心率加快,伴有延髓腹侧面头端胆碱酯酶活性降低和脊髓蛛网膜下腔灌流液中P物质样免疫反应活性升高。在延髓腹侧面头端应用阿托品或脊髓蛛网膜下腔注射P物质拮抗剂D-脯~2,D-苯丙~7,D-色~9-P物质均可阻断毒扁豆碱的心血管效应。脊髓蛛网膜下腔注射P物质抗血清或辣椒素均可减弱毒扁豆碱的升压反应。实验结果提示,毒扁豆碱作用于延髓腹侧面头端的M受体,兴奋了延髓-脊髓P物质能神经元下行通路,使之释放P物质,引起交感肾上腺髓质系统兴奋,从而使血压升高和心率加快。     

莱菔硫烷对人膀胱癌细胞周期的影响及机制研究

背景与目的:探讨莱菔硫烷对人膀胱癌细胞(T24)细胞周期的影响及作用机制。材料与方法:采用噻唑蓝(MTT)法研究不同浓度的莱菔硫烷对T24细胞生长的抑制作用并测定其半数抑制浓度(IC50);采用流式细胞仪检测莱菔硫烷对T24细胞周期的影响;采用westernblot研究莱菔硫烷对T24细胞中细胞周期蛋白依赖激酶抑制剂P16和P27的表达情况。结果:在较低剂量范围内(≤40μmol/L),随着作用剂量的增加,莱菔硫烷对T24细胞的生长的抑制作用也明显增强。10、20、40μmol/L莱菔硫烷的抑制率分别为(12.5±3.95)%,(25.0±2.50)%、(50.0±5.33)%;在较高剂量(60μmol/L~160μmol/L)时,这种抑制作用不再呈剂量依赖性;莱菔硫烷作用72h后的IC50值为(51.12±7.10)μmol/L;莱菔硫烷能使T24细胞周期阻滞于G0/G1期,20μmol/L莱菔硫烷作用48h后,在G0/G1峰之前出现凋亡峰;20μmol/L莱菔硫烷作用于T24细胞8、12、24h后能明显诱导P27蛋白的表达,作用早期(8h)时能诱导P16蛋白的表达。结论:莱菔硫烷能抑制T24细胞生长并使该细胞周期阻断在G0/G1期,其作用机制主要是通过诱导P27蛋白及早期诱导P16蛋白来实现的。     

Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile]), and verapamil N-demethylation (formation of norverapamil [2,8-bis(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (111) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18.In human liver microsomes (n=4), the intrinsic clearance (CL_int), as derived from the ratio of V _max/K_m, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9±1.0 vs 2.4±0.6 ml^*min^{-1 *}g^-1 mean±SD; p<0.05), S-Verapamil (16.8±3.3 vs 2.2±1.2 ml^* mini^*g^-1, p<0.05) and R-verapamil (12.1±2.9 vs 3.6 ±1.3 ml^*min^{-1 *} g^-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CL_int of D-703 formation in human liver microsomes showed a modest but significant degree of stereo selectivity (p<0.05) with a S/R-ratio of 1.41±0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5±4.5% and 45%, that of D-702 by 52.7±7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703.In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.     

Effect of Omeprazole on Diazepam Disposition in the Rat: in Vitro and in Vivo Studies

Purpose. The inhibitory effects of omeprazole on diazepam metabolism in vitro and in vivo are compared in the rat. Methods. 3-hydroxylation and N-demethylation of diazepam was investigated in the presence of a range of omeprazole concentrations (2-500µM) in hepatic microsomes and hepatocytes. Zero order infusions together with matched bolus doses of omeprazole were used to achieve a range of steady state plasma concentrations (10-50mg/ L) and to study the diazepam-omeprazole interaction in vivo. Results. The 3-hydroxlation pathway was more prone to inhibition (K_Is 108 ± 30 and 28 ± 11 µM in microsomes and hepatocytes, respectively) than the demethylation pathway (K_Is of 226 ± 76 and 59 ± 27 µM in microsomes and hepatocytes, respectively). In both in vitro systems, the mechanism of inhibition was competitive with Km/K_I ratios larger than 1 for the 3HDZ pathway and smaller than 1 for the NDZ pathway. There was an omeprazole concentration dependent decrease in diazepam clearance in vivo which could be modelled using a simple inhibition equation with a K_I of 57µM (19.8mg/L). In contrast there was no statistically significant change in the steady state volume of distribution for diazepam in the presence of omeprazole. Conclusions. The in vivo K_I for the omeprazole: diazepam inhibition interaction shows closer agreement with the K_I values obtained in hepatocytes than with those observed in microsomes.     

Cognition in relapsing-remitting multiple sclerosis: a multichannel event-related potential (P300) study

Auditory event-related potentials (AERP) were elicited in 47 patients with relapsing-remitting (RR) multiple sclerosis (MS) and 24 age-matched controls. MS patients had significantly prolonged N2 and P3 latencies as well as low P3 amplitude compared with controls. Seven of them exceeded 3 standard deviations from the control mean values. The observed N2 and P3 alterations are associated with the patients' disability status as it is defined by the Kurtzke expanded disability status scale (EDSS), but are not related to the duration of the disease. A possible cognitive decline as reflected in the observed AERP components alterations in MS patients is subsequently discussed.     

胃癌组织中KAI1、nm23及P53的表达及其临床意义

目的:探讨正常胃黏膜、不典型增生胃黏膜及癌组织中KAI1、nm23及P53蛋白的表达．方法:应用SP法免疫组化检测22例正常胃黏膜,65例不典型增生胃黏膜及74N胃癌组织中的KAI1、nm23及P53蛋白的表达．结果:正常胃黏膜、不典型增生胃黏膜及胃癌组织中,KAI1和nm23阳性率呈降低趋势,组间差异性有统计学意义(x2=20．885, P<0．001;x2=29．133,P<0．05):P53蛋白阳性表达率呈增加趋势,组间差异性有统计学意义(x2=21．954,P<0．001)．Fisher精确概率检验显示:在胃癌组中不同的浸润深度、有无淋巴结转移和脉管侵犯组内KAI1、nm23及 P53组阳性表达率的差异性有统计学意义(x2 =20．885,P<0．001;x2=29．133,P<0．05;x2= 21．954,P<0．001);而在年龄、性别组间的差异性无统计学意义．Spearman等级相关分析显示 KAI1与nm23表达呈正相关(r=0．859,P<0．05); KAI1与P53表达呈负相关(r=-0．859,P<0．05), nm23与P53表达呈负相关(r=-0．874,P<0．05) 结论:抑癌基因KAI1与nm23的缺失以及P53 蛋白的过表达可能是胃癌发生、发展及浸润和转移的重要原因之一．     

腺病毒介导的P16／CDKN2基因对TJ899细胞系活性的影响

（1）目的 研究5型腺病毒载体（Ad5)携带P16基因对恶性脑胶质瘤细胞系TJ899生长状态的影响。（2）方法 免疫组化（SP法）测定P16蛋白表达，MTT（methly thiazolyl tetrazolium,MTT)法测定恶性脑胶质瘤细胞系生长状态，克隆形成实验。（3）结果 重组体腺病毒能介导P16外源基因在恶性脑胶质瘤细胞系TJ899细胞中阳性表达，6d时肿瘤细胞生长抑制率为93%，并且能显地抑制肿瘤细胞的克隆形成能力。（4）结论 腺病毒介导P16基因能在肿瘤细胞中表达。并能明显抑制肿瘤细胞生长的状态。     

Electrophysiological Signs of Neurocognitive Deficits in Long-Term Leukemia Survivors

The long-term effects of disease and treatment on electrophysiologicalmeasures of neurocognitive function were studied in childrenwho had survived acute lymphoblastic leukemia (ALL) for at least4 years and were currently in remission. We report here changesin cognitive processing time as shown by the latency of theP3 wave of the auditory event-related EEG potential (ERP). P3latency was significantly prolonged in long-term ALL surivors,as well as in patients successfully trreated for solid tumors(ST)outside the CNS who received similar chemotherapy but did notreceive prophylactic treatment to the CNS. P3 latencies werestrongly correlated with measures of school performance andIQ in these individuals. The similarity in P3 latency betweenthe ALL and ST groups suggests that the treatments used on thesepateints produce changes in electrophysiological responses thatare associated with mild, but significant, cognitive deficits.     

Substance P induces inositol 1,4,5-trisphosphate and intracellular free calcium increase in cultured normal human epidermal keratinocytes

Abstract Substance P is a neuropeptide which is present in peripheral C nerve endings and released from them. Free nerve endings of C nerve are present in human epidermis. The effects of substance P on the transmembrane signaling system of pig epidermal sheets were previously reported. In these studies, a small amount of cells other than keratinocytes contaminated the epidermal sheets and the species difference from human was also noticed. Therefore we investigated the effects of substance P on cultured normal human epidermal keratinocytes. Alteration of intracellular free calcium (Ca²⁺) in single living keratinocytes was studied using an inverted fluorescence microscope and Ca²⁺ -sensitive dye, Fura 2-AM. Treatment of normal human epidermal kertinocytes with substance P resulted in an increase in inositol 1,4,5-trisphosphate and in intracellular Ca²⁺. Substance P inhibited DNA synthesis of the keratinocytes in a dose-dependent manner. These results are consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate hydrolysis of human keratinocytes, resulting in inositol 1,4,5-trisphosphate-Ca²⁺ signal.     

Increased reactivity to chemical but not to heat noxious stimuli in mice producing anti-idiotypic antibodies for substance P

Mice immunized against anti-substance P (anti-SP) monoclonal antibodies produced anti-SP anti-idiotypic antibodies (SPAb2). In a previous report. SPAb2 antibodies were found to have in vitro biological activity i.e. to behave either as agonists or as antagonists for substance P (SP) depending on the biological test. In this study, the involvement of SPAb2 in vivo biological activity has been tested. Because of the possible implication of SP in the generation and transmission of nociceptive information, we have tested the responsiveness of SPAb2 responding mice in behavioral nociceptive tests. SPAb2 mice showed very small behavioral variations in the hot plate test as compared with a control group of mice immunized against an unrelated monoclonal antibody. In the formalin test, however, SPAb2 mice displayed a significant increase in paw licking time, which was significantly correleted with SPAb2 serum concentration. These results are discussed in terms of the use of SPAb2 as pharmacological tools for studying the biological properties of SP receptors and more generally of auto anti-idiotypic antibodies in modulating behavior responses.