小鼠Usp25基因的雄激素反应元件的克隆与功能鉴定

目的我们前期研究中进行全基因组表达谱检测发现在雄激素受体（androgen receptor,Ar）基因睾丸支持细胞特异性敲除小鼠（S-Ar^-/y）睾丸组织中泛素特异性蛋白酶25（ubiquitin specific peptidase 25,usp25）基因表达较低。本研究的目的是了解雄激素及其受体是否可以作用于Usp25基因,并测定其雄激素反应元件（androgen-responsive element,ARE）。方法采用RT-qPCR方法检测Usp25基因表达量。通过生物信息学预测脚25基因上游可能的ARE,构建Usp25基因ARE报告质粒pGLP／Usp25。在TM4细胞中,采用荧光素酶报告系统分析雄激素及其受体对Usp25基因ARE活性的调控作用。结果在S-Ar^-/y小鼠睾丸组织中矾p25基因表达量比在野生型小鼠中显著降低。在TM4细胞中睾酮可以显著提高Usp25基因表达量。在TM4细胞中,雄激素可以显著提高pGLP／Usp25的荧光素酶活性。结论Usp25基因表达可以被雄激素睾酮激活,Usp25基因第一内含子519至1102bp区域含有ARE,可以调控启动子的转录水平。     

Expressions of genes related to genome stability and DNA repair in nasopharyngeal carcinoma clustering families

Objective： The aim of the study was to observe the expressions of genes related to genome stability and DNA repair in the members of nasopharyngeal carcinoma （NPC） clustedng families. Methods： In the Zhongshan City where there is highly incidence rate of NPC, we chose the members of the NPC clustering families as objects, and the patients of nasopharyngitis and NPC as the control group. We isolated the RNA from the nasopharyngeal tissue, and synthesized its cRNA, the genome stability and DNA repair genes chip technique, chemiluminescent detection and real-time fluorescence quantita- tive technique were used to examine the genome stability and DNA repair genes in the nasopharyngeal tissue. Results： More genome stability and DNA repair genes were up-regulated in the members of the NPC clustering families than the NPC patients, and the range of up-regulated was high, with the over up-regulated 100 times genes including TEP1, MSH4, PMS2LI. Fewer genome stability and DNA repair genes were down-regulated in the members of the NPC clustering families than the NPC patients, the ubiquitin genes almost were down-regulated, the results also could be confirmed by real-time fluorescence quantitative PCR. Conclusion： There are specially expression character of genome stability and DNA repair genes in the members of NPC clustering families.     

泛素蛋白酶体途径在急性早幼粒细胞白血病中作用的研究进展简

急性早幼粒细胞白血病（acute promyelocytic leukemia,APL）以表达PML-RARα为特征.泛素-蛋白酶体途径在全反式维甲酸及三氧化二砷诱导其降解过程中发挥重要作用,并参与细胞周期调节、转录调控等过程.深入研究该通路在APL中的作用有助于明确部分药物的起效机制及开拓APL的临床治疗思路.本文就泛素-蛋白酶体途径的组成、泛素-蛋白酶体途径介导的蛋白质修饰在APL中的作用、泛素化途径相关药物在APL中的应用进行综述.     

胃转流术对非肥胖型2型糖尿病大鼠骨骼肌组织中MG53蛋白表达的影响

目的研究胃转流术对非肥胖型2型糖尿病(T2DM)大鼠骨骼肌组织中E3泛素连接酶—mitsugumin53(MG53)蛋白及其mRNA,以及胰岛素受体底物1(IRS-1)mRNA表达的影响,探讨胃转流术改善骨骼肌胰岛素抵抗的可能机制。方法将24只雄性GK大鼠随机分为糖尿病手术组、糖尿病假手术组及糖尿病对照组,每组8只,若大鼠死亡,则重新制备模型补充;另外以8只Wistar大鼠作为正常对照组。应用Westem blot技术检测4组大鼠术后第8周时骨骼肌组织中MG53蛋白的表达水平,应用RT-PCR技术检测4组大鼠术后第8周时骨骼肌组织中IRS-1 mRNA和MG53 mRNA的表达水平。结果①糖尿病假手术组及糖尿病对照组大鼠的MG53 mRNA及其蛋白的表达水平均高于正常对照组和糖尿病手术组(P<0.05),但糖尿病手术组和正常对照组间比较、糖尿病假手术组和糖尿病对照组间比较差异均无统计学意义(P>0.05)。②与正常对照组比较,糖尿病手术组、糖尿病假手术组及糖尿病对照组大鼠的IRS-1 mRNA的表达水平均较低(P<0.05),但糖尿病手术组、糖尿病假手术组及糖尿病对照组大鼠的IRS-1 mRNA的表达水平比较差异无统计学意义(P>0.05)。结论胃转流术降低了非肥胖型T2DM大鼠骨骼肌组织中IRS-1蛋白的E3泛素连接酶—MG53蛋白的表达水平,减少了IRS-1蛋白的泛素化降解,这可能是骨骼肌胰岛素信号通路中IRS-1蛋白表达上调的主要机制之一,从而改善了骨骼肌的胰岛素抵抗,降低了血糖。     

内质网应激在UCH-L1抑制剂诱导细胞凋亡中的作用

目的探讨内质网应激(ERS)在泛素羧基末端水解酶-1(UCH-L1)抑制剂导致的细胞损伤过程中的作用。方法采用50μmol/L的UCH-L1抑制剂处理人神经母细胞瘤SK-N-SH细胞。于处理前(对照组)和处理后不同时间点(2、4、6、12、24h),分别采用MTT比色法和Western blotting法检测细胞活力和ERS相关蛋白Bip/Grp78、Xbp-1、p-eIF2α及其促凋亡转录因子CHOP/Gadd153的表达。结果MTT比色法检测发现,50μmol/LUCH-L1抑制剂处理后4h,SK-N-SH细胞活力下降43.1%(P<0.05),且随处理时间的延长继续呈现显著下降趋势(P<0.01)。Western blotting检测显示,经50μmol/LUCH-L1抑制剂处理后,SK-N-SH细胞ERS相关蛋白Bip/Grp78、p-eIF2α和Xbp-1表达分别于处理后2、4、6h时显著增加;转录因子CHOP/Gadd153表达则在处理后2h和4h时略有增加,6h后达到高峰。结论在UCH-L1抑制剂诱导的细胞凋亡过程中,ERS是导致细胞损伤的路径之一。     

Identification and preliminary characterization of ubiquitin C terminal hydrolase 1 (UCHL1) as a biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage

By using two different approaches, ubiquitin C‐terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage (ASAH) and presumably other CNS damage and disease states. Appropriate antibodies and a sensitive ELISA were generated, and the release of UCHL1 into CSF was compared with that of pNF‐H and S100β in a cohort of 30 ASAH patients. Both UCHL1 and pNF‐H showed persistent release into CSF in almost all patients in the second week postaneurysmal rupture (AR), and S100β levels rapidly declined to baseline levels in 23 of 30 patients. Seven of thirty patients showed persistently elevated S100β levels over the first 5 days post‐AR and also had relatively higher levels of pNF‐H and UCHL1 higher compared with the rest. These patients proved to have very poor outcomes, with 6 of 7 expiring. Patients who did reduce S100β levels tended to have a better outcome if pNF‐H and UCHL1 levels were also lower, and elevated UCHL1 levels in the second week post‐AR were particularly predictive of poor outcome. Acute coordinated releases of large amounts of UCHL1, pNF‐H, and S100β in 16 of 30 patients were observed, suggesting sudden loss of brain tissues associated with secondary events. We conclude that measurement of the CSF levels of these proteins reveals details of ASAH progression and recovery and predicts patient outcome. © 2010 Wiley‐Liss, Inc.     

Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4–2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1^−/− with Ndfip1^+/+ mice exposed to Fe reveals that Ndfip1^−/− brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.     

Novel pharmacological agents in clinical development for solid tumours

For decades, cancer therapy has focused on DNA-directed mechanisms of cytotoxicity, utilising agents with limited efficacy and significant toxicity. Recent advances in tumour biology have elucidated the molecular pathways implicated in the pathogenesis and progression of cancers and have resulted in the discovery of a variety of novel molecular targets for therapeutic intervention. Promising novel agents targeting signal transduction pathways, cell cycle regulation, angiogenesis and apoptosis are in clinical testing and are discussed in this review.