PS1 Interaction with CHIP and regulate the cellular level of tumor suppressor PTEN

阿尔茨海默氏病(Alzheimerdisease，AD)是一种以渐进性记忆力丧失和认知障碍的神经退行性疾病，患者脑部有两个明显的病理学特征：即细胞内神经原纤维缠结(neurofibrillarytangle，NFT)和细胞外形成淀粉样斑（senileplaque，SP）。大量的研究结果表明：部分的AD由基因突变引起，并在家族内遗传，其临床症状表现为较早出现AD相关病理特征，因此又称做早发型家族性AD（early onsetfamilialFAD）；其中早老素1（Presenilin1，PS1）/早老素2（Presenilin2）的基因突变是造成早发家族性痴呆的主要原因。PS1是γ分泌酶的重要...Alzheimer Disease (AD) is a neurodegenerative disorder which is characterized clinically by progressive loss of memory and impairment of cognitive abilities. Neurofibrillary tangle (NFT) and senile plaque（SP）are the two hallmark pathological features found in the brains of AD patients. A subset of AD develops disease phenotypes in the early stages of patients and is inheritable within kindred...学位：理学博士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：B20032602

Transcriptional regulation of PEN-2, a key component of the γ-secretase complex, by CREB

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's P-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APP alpha and A beta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2

Breeding high-yield oil-producing strain to use cheap carbon source by UV induced protoplast mutagenesis

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,CAndIdA PArAPSIlOSIS)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(gC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/l、58.12%和10.32 g/l,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/l、49.44%和9.17 g/l,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。[Objective] We used UV induced protoplast mutagenesis to study breed a new high-yielding lipid-producing strain which could use cheap carbon source.[Methods] Get the 1.5% glusulase and 1.0% cellulose solution.Hydrolyze to remove the cell wall and obtain the protoplast of 2A00015(Candida parapsilosis).Put it under the ultraviolet lamp for mutagenesis and cultivate regenrated wall.Then screen to get the high oil generated yeast which could be fermented by low-cost carbon source.Determine its fat acid components by gas chromatography with mass spectrometric(GC-MS).[Results] Cultivated the mutant strain with best mutation 2A00015/25 in the glucose.We found that the biomass, oil yield rate and oil production are separately 17.77 g/L, 58.12% and 10.32 g/L, which are separately 12.45%, 23.32% and 38.68% higher than the original strain.We have also cultivated the mutant strain in the waste molasses and found the biomass, oil yield rate and oil production are separately 18.54 g/L, 49.44% and 9.17 g/L, which are separately 9.09%, 21.16% and 32.18% higher than the original strain.The oil yield rate is lower in the waste molasses cultivation than that in glucose cultivation.However, in consideration of environment protection and cost of raw materials, the waste molasses is of much more advantages.It is tested that the fat generated from the waste molasses fermentation consists of eight kinds of fat acid.Its fat acid components are similar to the vegetable oil, in which the content of unsaturated fatty acid comprised 82.4% of the total fatty acid.[Conclusion] Basing on UV induced protoplast mutagenesis, we have successfully bred a new high-yielding oil strain which can make use of the low-cost carbon source, with its oil production rate 49.4% which has increased by 21.2%.深海(微)生物资源勘探与资源潜力评价项目(No.DY125-15-R-01

Y型聚乙二醇干扰素琢-2b注射液治疗HCV基因2/3型慢性丙型肝炎患者疗效和安全性的多中心随机对照试验研究

目的以标准剂量的聚乙二醇干扰素(Peg IFN)α-2a联合利巴韦林作为阳性对照,评价新型试验药物Y型Peg IFNα-2b注射液联合利巴韦林治疗2型/3型慢性丙型肝炎(CHC)患者的疗效和安全性。方法采用多中心、随机开放、阳性药对照的Ⅲ期临床试验,筛选符合要求的2型/3型CHC患者,按照2:1的比例随机分配到Y型Peg IFNα-2b组和Peg IFNα-2a组,同时口服利巴韦林,疗程24 w,停药随访24 w。采用Abbott Real Time HCV Genotype II检测HCV基因型,采用Cobas Taq Man实时定量PCR法检测血清HCV RNA水平。详细记录不良事件。主要疗效指标为持续病毒学应答(SVR),并进行非劣效检验。结果本试验实际入组2型/3型CHC患者255例,实际治疗241例。全分析集(FAS)数据显示,158例试验组和83例对照组患者SVR分别为85.4%(95%CI 79.94%~90.94%)和79.5%(95%CI 70.84%~88.20%,P=0.2402);对符合方案分析集(PPS)人群分析显示,试验组和对照组患者SVR分别为87.9%(95%CI 82.45%~93.27%)和85.9%(95%CI 77.82%~94.01%,P=0.7060),率差的95%可置信区间均符合非劣效标准;对PPS人群分析显示,85.8%受试者获得了早期病毒学应答(RVR),RVR的阳性预测值为90.1%;试验组和对照组不良事件发生率相似,分别为95.6%和95.2%,严重不良事件发生率分别为3.8%和3.6%。结论应用Peg IFNα联合利巴韦林治疗2型/3型CHC患者,新型试验药物Y型Peg IFNα-2b具有与对照药物Peg IFNα-2a相似的疗效和安全性。国家科技部“十二五”重大专项(编号:2012ZX10002-003)；“重大新药创制”十二五科技重大专项(编号:2012ZX09303019)

Prostratin Activates HIV-1 Transcription via nPKC-PKD3 Signaling Pathway

采用HIV-lTr-luC稳定细胞株分析其荧光素酶活性,研究PrOSTrATIn活化潜伏的艾滋病毒的分子机制.实验结果表明:随药物浓度升高和处理时间延长,PrOSTrATIn能明显激活HIV-1转录.采用PkC抑制剂拮抗nPkC活性,能有效阻断PrOSTrATIn对HIV-1的激活;反之,过表达nPkC能激活HIV-1转录.最后,敲低Pkd3高效拮抗PrOSTrATIn激活的HIV-1转录.因此,nPkC-Pkd3信号途径参与PrOSTrATIn活化HIV-1转录,从而激活潜伏的艾滋病毒.本研究为开发新的抗艾滋病药物筛选模型提供了可靠的理论依据.By employing HeLa cells with stable expression of HIV-LTR-Luciferase reporter gene,the underlying mechanism of HIV-1latency activated by prostratin treatment was disclosed.These results indicated that HIV-1transcription could be stimulated by prostratin treatment in a dose-and time-dependent manner.The prostratin-activated HIV-1transcription could be blocked by specific nPKC inhibitors,whereas overexpression of nPKCs could directly activate HIV-1transcription.Finally,HIV-1transcription induced by prostratin was blocked by knocking down of PKD3.Thus,nPKC-PKD3signaling pathway was engaged in prostratin-activated HIV-1transcription,which then released the latent HIV-1provirus.We proposed a new avenue for drug screening that was crucial to cure HIV-1infection.国家自然科学基金(81171192); 福建省自然科学基金(2013J01356

A Modified Method of QuikChange Site-Directed Mutagenesis

通讯作者: [email protected][中文文摘]DNA传统的快速点突变方法即"一步突变法"虽然可使DNA产生突变,但效率不高.市场上出售的快速点突变试剂盒,其突变效率可达80%以上,然而价钱昂贵.本文论述的快速点突变方法,在传统的"一步突变法"基础上做了改进,通过对引物突变点5′端序列Tm值的确定,使引物设计变得简单,初学者可轻松完成DNA的定点突变,而且大大降低了成本,适用于对大量DNA进行定点突变试验.试验结果表明:这种经过改良的方法突变率为96.22%,可为研究人员的试验带来便利,是一种值得推广的新方法.[英文文摘] traditional method that introduced mutations to DNA was the QuikChangeTM Site-Directed Mutagenesis,called"One-Step Site-Directed Mutagenesis".However,it had low efficiency.The QuikChangeTM Site-Directed Mutagenesis Kit could create mutants about 80% efficiency,but it was too expensive.The protocol in this paper was a modification of the traditional "One-Step Site-Directed Mutagenesis" and simplified the design of primers so that the DNA could be easily mutated by novice.Our results indicated that the efficiency of this modified method could reach 96. 22% .Th is protocolm aight be applied for a large number of DNA samples needed to bem utated due to its low co s.t So it is worth widely application.国家基础科学人才培养基金项目(J0630649);国家自然科学基金(30670408,30572077);福建省自然科学基金(2008J0108)资

Investigation and Comparison of Linear and Branched Polyethylenimine for Gene Delivery in Vitro

通讯作者: liurz@ xmu.edu.cn[中文文摘]聚乙烯亚胺(PEI)是一类新兴的阳离子多聚物非病毒载体,可缩聚DNA分子形成颗粒并转入真核细胞进行表达.本文选用分子量750 ku分枝状PEI与分子量25 ku线性PEI转染增强型绿色荧光蛋白(EGFP)报告基因于HeLa细胞,并对这两种PEI的转染效率进行比较.利用MTT法检测比较线性PEI与分枝状PEI的细胞毒性.通过凝胶阻滞实验比较两种PEI结合DNA能力.最后利用肝素介导释放实验比较两者在形成PEI/DNA复合物后释放DNA的能力.研究结果表明:线性PEI转染效率优于分枝状PEI,且随PEI/DNA质量比的增加而升高,而分枝状PEI则相反;两种PEI细胞毒性在一定范围内相近;分枝状PEI结合DNA能力略强于线性PEI;但是分枝状PEI释放DNA的能力远小于线性PEI.这可能导致DNA在细胞内无法从PEI/DNA复合体上释放出来,从而影响转录的正常进行,最终导致分枝状PEI转染效率下降.［英文文摘］Polyethylenimine is a widely-used non-viral vector with polycation.It can condense DNA to form PEI/DNA complexes,which can be transfected into mammalian cells for gene expression.In this study,enhanced green fluorescence protein was transfected into HeLa cells by linear PEI and branched PEI,then compared the cytotoxic effects by the method of MTT assay.The ability of DNA binding of both linear PEI and branched PEI was identified,and the level of DNA release from PEI/DNA complexes was compared.The results showed that the　transfection efficiency of linear PE Iwasm uch better than that of branched PEI. Linear PEI transfection　efficiency increased with increasing　PEI /DNA ratio, while branched PEI　was contrary. Both PEI have the same cytotoxic effects in selected range and they have amlost the same ability of　DNA binding. In addition, DNA ismuchm ore difficult to release from bran ched-PEI/DNA comp lexes than from linear-PE I/DNA complexes, which　may be the reason of the poor transcription leading to decreasing transfection efficiency of branched PEI.国家基础科学人才培养基金项目(J0630649); 国家自然科学基金(30670408,30572077); 福建省自然科学基金(2008J0108)资

Method for Fluorescent Quantification of Cellular Green Fluorescence Protein and Its Application

通讯作者: [email protected][中文文摘]绿色荧光蛋白是在生化与分子生物学研究领域中普遍使用的一种基因表达报告系统显色物.对于细胞内绿色荧光蛋白表达水平的定量检测,目前尚缺乏一套准确?简便?易行的技术.本文以增强型绿色荧光蛋白(EGFP)为报告基因,用分子量为25 ku的线性聚乙烯亚胺介导进入HeLa细胞并表达.利用多功能酶标仪测定细胞裂解液的荧光强度,结果显示:这种新方法测定的荧光强度能很好反映细胞中EGFP的蛋白表达水平,并在短时间内完成对EGFP表达水平的定量检测.该方法简单有效,且不依赖于大型仪器设备,具有较高的准确性与灵敏度,在实际应用中简便可靠,具有推广应用的意义.[英文文摘]Green Fluorescence Protein(GFP),a material of reporter gene expression system,was widely utilized in the different fields of biological research.However,it is difficult to quantitatively measure the GFP level of cells in a simple and effective way.In this study,we set up an easy,sensitive and efficient method.Enhanced Green Fluorescence Protein(EGFP) was transfected into HeLa cells by 25 ku linear polyethylenimine.After preparation of cell lysate,the fluorescence intensity of expressed EGFP can be detected by mu ltifunctionalm icroplate reader without the utilization of expensive apparatus.Our results indicated that thenew method is highly stable and precise in application and allows sensitive detection of prote in level in HeLa cells. It has been well proved that the fluorescence in tensity of EGFP is consistent with the protein level in cells.国家基础科学人才培养基金项目(J0630649);国家自然科学基金(30670408,30572077);福建省自然科学基金(2008J0108)资

Divalent cation tolerance protein binds to beta-secretase and inhibits the processing of amyloid precursor protein

National Natural Science Foundation of China [81171192]; XMU Basic Training Program of Undergraduate [CXB2011019]; Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering of Xiamen University [201101]The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by beta-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and gamma-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1-interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease

Presenilins regulate the cellular level of the tumor suppressor PTEN

Alzheimer's Disease (AD) is characterized by amyloid plaques consisting of beta-amyloid (A beta) peptides and neurofibrillary tangles consisting of hyperphosphorylated tau protein. A beta is proteolytically derived from its precursor protein through cleavages by beta-secretase and beta-secretase complex comprising presenilins (PS, PS1/PS2), nicastrin, APH-1 and PEN-2. PS1 is also known to activate the PI3K/Akt cell survival pathway in ay-secretase-independent manner. The tumor suppressor PTEN, which antagonizes the PI3K/Akt pathway, has increasingly been recognized to play a key role in neural functions and its level found reduced in AD brains. Here, we demonstrate that the protein level of PTEN is dramatically reduced in cultured cells and embryonic tissues deficient in PS, and in the cortical neurons of PS1/PS2 conditional double knockout mice. Restoration of PS in PS-deficient cells reverses the reduction of PTEN. Regulation of PTEN by PS is independent of the PS/gamma-secretase activity since impaired gamma-secretase by the gamma-secretase inhibitor treatment or due to nicastrin deficiency has little effect on the protein level of PTEN. Our data suggest an important role for PS in signaling pathways involving PI3K/Akt and PTEN that are crucial for physiological functions and the pathogenesis of multiple diseases. (c) 2006 Elsevier Inc. All rights reserved