泛素C末端水解酶L1的生理和病理学意义

泛素C末端水解酶L1(ubiquitin carboxy-terminal hydrolases L1)属于泛素C末端水解酶家族成员,但是泛素C末端水解酶L1酶活性非常特异,不仅具有泛素C末端水解酶活性,而且具有泛素C末端聚合酶的活性.因此,泛素C末端水解酶L1,不仅在泛素化蛋白降解途径中起到关键的作用,也在其他的泛素信号途径,如在K63-多聚泛素信号途径中起重要的作用.由于泛素C末端水解酶L1特异的蛋白酶活性,也赋予了泛素C末端水解酶L1多种生物学功能,在神经发育发生、精子发生、卵子发生和受精等方面有着重要的作用.泛素C末端水解酶L1突变也与帕金森症等神经元退化疾病紧密相关.泛素C末端水解酶L1在甲状腺、肺等多种组织的超表达,也与该组织的癌症发生有着密切的联系.     

APP/PS1转基因小鼠海马结构UCH-L1的表达及亚甲蓝对其改变的影响

目的探讨亚甲蓝(MB)对APP/PS1转基因小鼠记忆相关蛋白泛素羧基末端水解酶1(carboxyl-terminalhydrolase-L1,UCH-L1)在海马结构的表达及记忆改善的影响。方法 3月龄APP/PS1转基因小鼠及相同品系的野生小鼠分为3组,每组10只:治疗组,APP/PS1小鼠口服亚甲蓝(25mg/kg/d)4个月;模型组,APP/PS1小鼠无药物干预;对照组为正常野生小鼠。待三组小鼠均为7月龄时,跳台实验测试三组小鼠的学习记忆能力;Western blot及免疫荧光技术检测海马结构UCH-L1的含量变化。结果亚甲蓝可以减少小鼠跳台试验错误次数,延长小鼠跳台试验的潜伏期(P<0.01)。亚甲蓝治疗组海马结构的可溶性UCH-L1含量明显增多(P<0.01)。结论亚甲蓝可能是通过上调海马结构可溶性UCH-L1的表达改善APP/PS1小鼠的学习记忆能力。     

多巴胺脂质体经鼻给药治疗帕金森氏病的研究

目的:研制多巴胺脂质体,用于治疗帕金森氏病。方法:用大豆卵磷脂和油酸制备多巴胺脂质体;采用昆明种小白鼠,腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)建立帕金森氏病动物模型,用于评价多巴胺脂质体经鼻给药的治疗帕金森氏病的疗效。结果:多巴胺脂质体的平均粒径为20-120nm,Zeta电位为-25.6m V,对多巴胺的包封率为(98.2±1.6)%(n=3);经鼻给药,多巴胺脂质体具有显著的抗帕金森氏病作用(p0.05)。结论:采用大豆卵磷脂、油酸可以制备出多巴胺脂质体,经鼻给药用于治疗帕金森氏病有效。     

UCH-L1在乳腺癌中的表达及其与转移关系的研究

目的研究泛素羧基末端水解酶L1(UCH-L1)与磷酸化p38(p-p38)在乳腺癌组织、细胞系中的表达情况、两种蛋白的表达与临床病理特征的关系以及UCH-L1与乳腺癌侵袭转移的关系。方法用免疫组织化学方法检测乳腺癌组织中UCH-L1与p-p38蛋白的表达情况,用Western Blot方法检测乳腺癌组织以及细胞系中UCH-L1与p-p38蛋白的表达情况。应用UCH-L1特异性抑制剂作用于乳腺癌高侵袭高转移细胞系MDA-MB-435s后,用Western Blot观察UCH-L1与p-p38蛋白表达改变的情况,用Transwell实验检测MDA-MB-435s细胞侵袭潜能的改变。结果 UCH-L1和p-p38蛋白在乳腺浸润性导管癌中的表达高于其在癌旁正常乳腺组织中的表达(P=0.012,P=0.001),二者呈正相关(r=0.397,P=0.000),并与乳腺癌的TNM分期(P=0.017,P=0.010)、淋巴结转移情况(P=0.033,P=0.021)相关。乳腺上皮细胞系MCF-10A、乳腺癌低侵袭低转移细胞系MCF-7和乳腺癌高侵袭高转移细胞系MDA-MB-435s中两种蛋白表达水平呈递增趋势(P均<0.05)。UCH-L1特异性抑制剂可以浓度依赖性地下调MDA-MB-435s细胞系中p-p38蛋白的表达水平(P均<0.05),并能抑制乳腺癌细胞的侵袭转移潜能。结论 UCH-L1、p-p38过表达与乳腺癌的TMN分期、淋巴结转移有关。UCH-L1可能通过上调p-p38介导乳腺癌转移。     

泛素C端水解酶L1的RNA干扰载体的构建及效果评价

目的:获得抑制效果好的泛素 C 端水解酶 L1(UCH-L1)基小干扰 RNA(siRNA)干扰载体.方法:根据 Gen?Bank 中大鼠 UCH-L1基序列设计并合成4对 siRNA 寡核苷酸序列,将4对寡核苷酸序列退火成双链后分别插入siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR 中构建4个 siRNA 表达质粒,测序鉴定后将4个 siRNA 表达质粒分别转染 HEK293细胞,利用 Western 印迹和 qPCR 方法检测干扰效果;将干扰效果最好的质粒包装成腺病毒,感染大鼠血管平滑肌细胞(VSMC),并采用 TNFα干预诱导 UCH-L1表达升高,Western 印迹验证干扰效果.结果:测序分析证实4对 siRNA 寡核苷酸序列分别插入 siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR;qPCR 检测与 Western 印迹均表明第3号 siRNA 表达载体对 UCH-L1表达的抑制程度最高,将其包装成腺病毒并转染 VSMC 能显著抑制 TNFα诱导的UCH-L1表达升高.结论:构建并筛选出干扰效果好的 UCH-L1 siRNA 干扰载体.     

生物标记物对脑损伤患儿早期诊断及监测价值的研究进展

脑损伤是新生儿期危害严重的疾病之一,可导致脑瘫、运动发育迟缓、认知功能障碍及学习困难等后遗症,严重影响了新生儿的健康发育及生活质量的提高。新生儿脑损伤(NBI)是一种由多种原因导致的范围很广的疾病,其临床表现缺乏特异性,临床上在判断其损伤严重程度、持续时间及产前损伤时间等存在较大的困难,受到广大科研者及临床医师的重视。目前,影像学方法是NBI确诊的主要手段,但影像学检查通常存在滞后性和一定的局限性。体液生物标记物水平在脑损伤后会较早发生变化,通过检测其水平变化可早期预测脑损伤情况。近年来,新生儿各种体液中已检测出多种具有敏感性的脑损伤生物标记物,主要包括神经元特异性烯醇化酶(NSE)、泛素羟基末端水解酶L-1(UCH-L1)、S100B蛋白、Tau蛋白、髓鞘碱性蛋白(MBP)、胶质纤维酸性蛋白(GFAP)、激活素A等,本研究对上述常用生物标记物在NBI中的应用情况以及研究进展进行综述,探讨其临床应用前景。     

人神经生长因子的原核高效表达

神经生长因子(nerve growth factor,NGF)是神经系统最重要的生物活性分子之一,也是最早发现和最典型的神经营养因子。它影响外周和中枢神经系统某些神经元的存活与分化;NGF在神经损伤时可保护其效应神经元,促进神经纤维再生,增加脑移植中某些神经元的存活;它对外伤、中毒、老化等因素引起的脑疾患有治疗作用,并具有神经修复功能,尤其是对早老性痴呆症、帕金森氏病的治疗作用比较乐观。虽然NGF在神经损伤和神经退行性病变的诊断和治疗中有巨大的临床应用价值,但天然NGF受到雄性小鼠颌下腺这     

细胞周期蛋白依赖性蛋白激酶5在中枢神经系统发育和神经退行性疾病中的作用

脯氨酸引导的丝/苏氨酸蛋白酶——细胞周期蛋白依赖性蛋白激酶5(cyclin-dependent kinase5,Cdk5)是细胞周期素依赖的蛋白酶家族中一个特殊成员。Cdk5不参与细胞周期调控,其活化需要与神经元内广泛表达的激活因子——p35或p39相结合。正常情况下,Cdk5的转录及活性受到体内相关机制的严格调控。在神经系统发育及成熟阶段,Cdk5通过磷酸化细胞骨架蛋白、信号分子以及调节蛋白等众多底物蛋白的特异性丝/苏氨酸位点,在神经元的迁移分化、存活和突触的发生、信息传递、可塑性等诸多方面发挥重要的作用。此外,在一些病理条件下,p35的病理性剪切和Cdk5/p25的形成所导致的Cdk5活性失调及其亚细胞分布改变则促进了神经元的凋亡或死亡,参与了阿尔茨海默氏病(Alzheimer's disease,AD)、帕金森氏病(Parkinson’s disease,PD)、亨廷顿氏病(Huntington’s disease,HD)以及脊髓侧索硬化症(amyotrophiclateralsclerosis,ALS)等众多神经退行性疾病的发生发展过程。本文综述了Cdk5在中枢神经系统发育和神经退行性疾病中的作用研究方面的进展。     

MPTP与帕金森氏症

1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)是一种神经毒,在人和灵长类动物,它选择性损害黑质纹体系统多巴胺神经元,使多巴胺(DA)及其代谢产物降低,引起典型的帕金森氏样症状。MPTP类毒性物质有其构效关系,其毒性主要与在脑内形成1-甲基-4-苯吡啶(MPP~ )有关。正常存在于脑内的色氨衍生物2-N-甲基四氢β-卡啉(2M-THBC),其构造及作用均类似于MPTP样物质,由此可以推断,MPTP除作为部分帕金森氏症的直接诱发原因外,极有可能是揭示原因不明的原发性帕金森氏病发病机理的一条重要途径。     

骨钙素的中枢调控作用及其生物学机制

骨钙素(OCN)能调节多种外周组织器官的生理结构与功能,也发挥重要的中枢调控作用,与个体的学习和记忆等高级认知功能密切相关。研究表明,OCN穿过血脑屏障进入大脑,并与神经元或神经胶质细胞膜上的G蛋白偶联受体(GPCR)家族成员GPR158和GPR37结合,激活或抑制细胞内相关信号通路,改变神经元或神经胶质细胞的生理活性。OCN在脑内的作用主要包括调节5-羟色胺、多巴胺、去甲肾上腺素和γ-氨基丁酸等神经递质合成与释放、增加脑源性神经营养因子表达、促进海马神经发生、增强海马神经元自噬及维持髓鞘稳态等。此外,OCN还能参与调控多种神经退行性疾病的病理生理学进程。在阿尔茨海默病(AD)中,OCN干预能够部分减少β-淀粉样蛋白(Aβ)沉积及Aβ诱发的细胞毒性等,改善学习和记忆能力缺陷;在帕金森氏病(PD)中,OCN干预能够部分抑制黑质和纹状体多巴胺能神经元丢失,增加酪氨酸羟化酶含量及降低神经炎症等,缓解运动功能障碍。本文通过解析GPR158和GPR37的结构与功能,分析OCN在脑内的作用及其生物学机制,探讨OCN对AD和PD等神经退行性疾病的影响,为进一步筛选促进脑健康的新型靶点提供依据。     

Salvianolic acid inhibits the effects of high glucose on vascular endothelial dysfunction by modulating the Sirt1‐eNOS pathway

Salvianolic acid (SA) is known for improving blood circulation, scavenging hydroxyl radicals, and preventing platelet aggregation. The research explored whether SA can protect against cardiovascular disease induced by high glucose conditions. Our results indicate that SA significantly increases cells viability and nitric oxide levels while decreasing reactive oxygen species generation. SA upregulated the expression levels of Bcl‐2 and decreased the levels of Bax, cleaved caspase‐3, and cleaved caspase‐9. Furthermore, the expression levels of Sirtuin 1 (Sirt1) and p‐endothelial nitric oxide synthase (eNOS) were markedly increased in response to SA treatment. Moreover, exposure of human umbilical vein endothelial cells to Ex527 resulted in reducing expression of p‐eNOS. However, the beneficial effects of SA were abolished partially when Ex527 was added. These findings suggest that SA can be used as a potential therapeutic to protect against high glucose‐induced endothelial injury by modulating Sirt1‐eNOS pathway.     

联合检测血清NGAL、KIM-1和SCr可有效提高慢性肾病分期诊断

为了探讨中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)和肾损伤分子-1 (kidney injury molecule-1, KIM-1)以及血肌酐(serum creatinine, SCr)联合检测对慢性肾病(chronic kidney disease, CKD)的早期诊断价值,本研究收集260例肾病患者和85例健康体检者,检测其血清NGAL、KIM-1和SCr水平。依据肾功能分级标准,CKD患者分为CKD 1期(53例),CKD 2期(68例),CKD 3期(71例),CKD 4期(46例)和CKD 5期(22例),并分析以上指标在各组间的含量差异,及其联合测定对CKD早期的敏感性。与健康对照组相比较,CKD 1期、CKD 2期、3期、4期和5期患者的NGAL、KIM-1水平均明显升高(p<0.001)。血清SCr含量在CKD 3期、4期和5期组较健康对照组显著增加(p<0.001)。以上3项指标均随着CKD严重程度增加而升高。各组指标阳性率分析显示,3项联合检测阳性率高于单项检测阳性率。ROC曲线分析NGAL、KIM-1、SCr对CKD诊断的AUC值F分别是为0.824、0.805、0.856。相关性分析结果显示,GFR和NGAL、KIM-1、SCr相关系数分别是r=-0.784、-0.756、-0.728 (p<0.05)。NGAL与KIM-1、SCr的相关系数分别是r=0.932、0.764 (p<0.05);KIM-1与SCr的相关系数r分别是0.791 (p<0.05)。本研究初步得出结论:血清NGAL、Kim-1可作为CKD早期诊断的重要指标,联合检测血清NGAL、Kim-1、SCr可有效提高CKD早期肾损伤诊断的敏感度,对CKD的分期诊断和治疗具有极其重要的临床价值。     

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

All-trans-retinoic Acid Reduces BACE1 Expression under Inflammatory Conditions via Modulation of Nuclear Factor κB (NFκB) Signaling

Insulin resistance and neuroinflammation have emerged as two likely key contributors in the pathogenesis of Alzheimer disease (AD), especially in those sporadic AD cases compromised by diabetes or cardiovascular disease. Amyloid-β (Aβ) deposition and its associated inflammatory response are hallmarks in sporadic AD brains. Elevated expression and activity of β-secretase 1 (BACE1), the rate-limiting enzyme responsible for the β-cleavage of amyloid precursor proteins to Aβ peptides, are also observed in sporadic AD brains. Previous studies have suggested that there is therapeutic potential for retinoic acid in treating neurodegeneration based on decreased Aβ. Here we discovered that BACE1 expression is elevated in the brains of both Tg2576 transgenic mice and mice on high fat diets. These conditions are associated with a neuroinflammatory response. We found that administration of all-trans-retinoic acid (atRA) down-regulated the expression of BACE1 in the brains of Tg2576 mice and in mice fed a high fat diet. Moreover, in LPS-treated mice and cultured neurons, BACE1 expression was repressed by the addition of atRA, correlating with the anti-inflammatory efficacy of atRA. Mutations of the NFκB binding site in BACE1 promoter abolished the suppressive effect of atRA. Furthermore, atRA disrupted LPS-induced nuclear translocation of NFκB and its binding to BACE1 promoter as well as promoting the recruitment of the corepressor NCoR. Our findings indicate that atRA represses BACE1 gene expression under inflammatory conditions via the modulation of NFκB signaling.     

1-(Benzo[d]thiazol-2-yl)-3-phenylureas as dual inhibitors of casein kinase 1 and ABAD enzymes for treatment of neurodegenerative disorders

Several neurodegenerative disorders including Alzheimer’s disease (AD) have been connected with deregulation of casein kinase 1 (CK1) activity. Inhibition of CK1 therefore presents a potential therapeutic strategy against such pathologies. Recently, novel class of CK1-specific inhibitors with N-(benzo[d]thiazol-2-yl)-2-phenylacetamide structural scaffold has been discovered. 1-(benzo[d]thiazol-2-yl)-3-phenylureas, on the other hand, are known inhibitors amyloid-beta binding alcohol dehydrogenase (ABAD), an enzyme also involved in pathophysiology of AD. Based on their tight structural similarity, we decided to evaluate series of previously published benzothiazolylphenylureas, originally designed as ABAD inhibitors, for their inhibitory activity towards CK1. Several compounds were found to be submicromolar CK1 inhibitors. Moreover, two compounds were found to inhibit both, ABAD and CK1. Such dual-activity could be of advantage for AD treatment, as it would simultaneously target two distinct pathological processes involved in disease’s progression. Based on PAMPA testing both compounds were suggested to permeate the blood-brain barrier, which makes them, together with their unique dual activity, interesting lead compounds for further development.