泛素连接酶LNX1促进外源PBK的泛素化和降解

 目的 研究LNX1对其相互作用蛋白PBK的泛素化和降解。方法 克隆、原核表达、纯化了一系列重组人LNX1截断体蛋白和LNX1全长蛋白;在体外泛素化体系中研究其对PBK的泛素化,哺乳动物细胞内研究其对外源PBK的泛素化和降解。结果 在体外泛素化体系中LNX1泛素化PBK,并研究了不同LNX1截断体对PBK泛素化的影响;发现在哺乳动物细胞内外源LNX1促进外源PBK的泛素化,进而导致其通过蛋白酶体降解。结论 研究发现了LNX1对外源PBK的泛素化和降解,为研究LNX1的生理功能提供了重要线索。     

Defining in vivo dose-response curves for kidney DNA adduct formation of aristolochic acid I in rat,mouse and human by an in vitro and physiologically based kinetic modeling approach

Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.     

A physiologically based kinetic (PBK) model describing plasma concentrations of quercetin and its metabolites in rats

Biological activities of flavonoids in vivo are ultimately dependent on the systemic bioavailability of the aglycones as well as their metabolites. In the present study, a physiologically based kinetic (PBK) model was developed to predict plasma concentrations of the flavonoid quercetin and its metabolites and to tentatively identify the regiospecificity of the major circulating metabolites. The model was developed based on in vitro metabolic parameters and by fitting kinetic parameters to literature available in vivo data. Both exposure to quercetin aglycone and to quercetin-4′-O-glucoside, for which in vivo data were available, were simulated. The predicted plasma concentrations of different metabolites adequately matched literature reported plasma concentrations of these metabolites in rats exposed to 4′-O-glucoside. The bioavailability of aglycone was predicted to be very low ranging from 0.004%-0.1% at different oral doses of quercetin or quercetin-4′-O-glucoside. Glucuronidation was a crucial pathway that limited the bioavailability of the aglycone, with 95–99% of the dose being converted to monoglucuronides within 1.5–2.5 h at different dose levels ranging from 0.1 to 50 mg/kg bw quercetin or quercetin-4′-O-glucoside. The fast metabolic conversion to monoglucuronides allowed these metabolites to further conjugate to di- and tri-conjugates. The regiospecificity of major circulating metabolites was observed to be dose-dependent. As we still lack in vivo kinetic data for many flavonoids, the developed model has a great potential to be used as a platform to build PBK models for other flavonoids as well as to predict the kinetics of flavonoids in humans.     

肺及肺癌组织芯片PBK/TOPK蛋白表达：检测的可靠性

背景：组织芯片技术可高通量检测组织中的生物分子。但是组织芯片所用的组织很小,直径仅0.6 mm,且肿瘤细胞具有异质性,所以组织芯片检测结果是否可靠尚不清楚。 目的：比较石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK 蛋白在正常成人肺泡Ⅱ型上皮细胞、人胚胎肺泡上皮细胞、肺癌原发灶和相应的淋巴结转移灶的表达差异,探讨组织芯片检测PBK/TOPK蛋白表达的可靠性。 方法：采用组织微阵列技术按照正常成人肺组织,胚胎肺组织,肺鳞癌原发灶及其相应淋巴结转移癌,肺腺癌原发灶及其相应淋巴结转移癌,肺小细胞癌原发灶及其相应淋巴结转移癌,肺大细胞癌原发灶及其相应淋巴结转移癌的顺序依次构建包含760点阵的石蜡组织芯片。应用免疫组化SP法,检测石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK蛋白的表达。应用Leica Q500MC图像分析系统分别对石蜡组织芯片和常规石蜡切片上PBK/TOPK蛋白表达的阳性单位和阳性表达率进行定量测试。 结果与结论：组织芯片和对应常规切片相应细胞中PBK/TOPK 的阳性强度及阳性表达率基本相同,相对偏差不足0.6%,两者比较差异无显著性意义。结果说明石蜡组织芯片和常规组织切片检测PBK/TOPK蛋白表达结果高度一致;应用组织芯片检测肺癌、胚胎肺及正常肺组织中PBK/TOPK蛋白的表达结果可靠。 关键词：肺癌;组织芯片;PBK/TOPK;免疫组化;可靠性     

肺及肺癌组织芯片PBK/TOPK蛋白表达:检测的可靠性

背景:组织芯片技术可高通量检测组织中的生物分子.但是组织芯片所用的组织很小,直径仅0.6 mm,且肿瘤细胞具有异质性,所以组织芯片检测结果是否可靠尚不清楚.目的:比较石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK蛋白在正常成人肺泡Ⅱ型上皮细胞、人胚胎肺泡上皮细胞、肺癌原发灶和相应的淋巴结转移灶的表达差异,探讨组织芯片检测PBK/TOPK蛋白表达的可靠性.方法:采用组织微阵列技术按照正常成人肺组织,胚胎肺组织,肺鳞癌原发灶及其相应淋巴结转移癌,肺腺癌原发灶及其相应淋巴结转移癌,肺小细胞癌原发灶及其相应淋巴结转移癌,肺大细胞癌原发灶及其相应淋巴结转移癌的顺序依次构建包含760点阵的石蜡组织芯片.应用免疫组化SP法,检测石蜡组织芯片和对应组织常规石蜡切片PBK/TOPK蛋白的表达.应用Leica Q500MC图像分析系统分别对石蜡组织芯片和常规石蜡切片上PBK/TOPK蛋白表达的阳性单位和阳性表达率进行定量测试.结果与结论:组织芯片和对应常规切片相应细胞中PBK/TOPK的阳性强度及阳性表达率基本相同,相对偏差不足0.6%,两者比较差异无显著性意义.结果说明石蜡组织芯片和常规组织切片检测PBK/TOPK蛋白表达结果高度一致;应用组织芯片检测肺癌、胚胎肺及正常肺组织中PBK/TOPK蛋白的表达结果可靠.     

Expression of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) in human urinary bladder transitional cell carcinoma

The objective of this study was to evaluate the expression pattern of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) and its clinical significance in human bladder cancer (BC).     

B细胞非霍奇金淋巴瘤PBK/TOPK表达与增殖活性的关系

目的研究PBK／TOPK在反应性淋巴组织增生和B细胞非霍奇金淋巴瘤组织（B cell non—Hodgkin’s lymphoma,B—NHL）中的表达和肿瘤增殖活性的关系,探讨TOPK在淋巴瘤组织中发生发艮中的作用及其临床意义。方法运用SP法免疫组化法检测PBK／TOPK和Ki-67在B细胞性侵袭性淋巴瘤22例、惰性淋巴瘤21例、淋巴结反应性增生15例中的表达。结果TOPK在惰性淋巴瘤中的阳性率明显低于侵袭性淋巴瘤（P〈0．01）。Ki-67在反应性淋巴结增生和B—NHL中的表达与PBK／TOPK非常一致,二者存在正相关（P〈0．01）。在淋巴结反应性增牛中,PBK／TOPK主要表达于激活的中心母细胞,同时这些细胞亦表达Ki-67抗原。结论TOPK能激活B淋巴细胞,并与B—NHL的细胞增殖活性和恶性程度密切相关,提示TOPK在B细胞淋巴瘤的恶性增殖过程中发挥重要作用,为B细胞淋巴瘤的预后削断和诊治提供新的依据和靶点。     

PI3K/Akt信号转导通路与甲状腺结节

近年来研究发现,磷脂酰肌醇3激酶(PDK)/蛋白激酶B(Akt)信号转导通路在细胞的增殖、分化、生存和凋亡过程中起重要调节作用.结节性甲状腺肿、甲状腺腺瘤、甲状腺癌及桥本甲状腺炎病变中存在着PI3K/Akt信号转导通路的过度激活.PI3K/Akt信号转导通路的活化使甲状腺细胞增殖指数和生长指数明显升高,刺激甲状腺滤泡持续生长,促进甲状腺结节的发生、发展.研究此通路中相关分子的表达,对探索甲状腺结节的病因、发病机制和防治策略具有重要意义.     

PBK/TOPK在人乳腺及乳腺癌组织的表达及意义

目的:观察PBK/TOPK在人正常乳腺及乳腺癌组织的分布和表达,探讨作为肿瘤标志物的可能性.方法:应用免疫组织化学EnVision法,在含有人体20种正常组织及14种癌组织的组织芯片上进行PBK/TOPK 染色,检测PBK/TOPK 蛋白的表达.结果:在20种正常组织中,只有头皮的汗腺、睾丸的精原细胞、肝脏的胆管上皮、食管粘膜下腺体、胰腺外分泌部的导管上皮和肾脏的远曲小管7种组织有不同数量细胞明确阳性表达.在14种癌组织中,只有乳腺癌、肺癌、甲状腺癌3种癌组织中存在PBK/TOPK 的大量强阳性表达,而在正常乳腺组织不表达PBK/TOPK.结论:PBK/TOPK在正常组织的阴性表达及其相应恶性肿瘤的强阳性表达,为其成为研究肿瘤起源与发生的标志物提供了一定依据.     

PBK/TOPK通过改变糖酵解关键酶表达水平增加结直肠癌细胞放射敏感性

目的:探讨在结直肠癌细胞中PDZ结合激酶(PDZ binding kinase,PBK)/T-淋巴因子激活的杀伤细胞来源的蛋白激酶(T-cell-originated protein kinase,TOPK)能否通过影响糖酵解关键酶表达改变放射敏感性.方法:通过慢病毒转染敲低LS174T细胞中的PBK/TOPK表达水平...