Subcellular localization of PS1 based on PS1/GFP fusing protein

Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer’s disease (EOFAD). The fusion genes, GFPPS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT II (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution. __________ Translated from China Biotechnology, 2006, 26(6): 17-22 [译自: 中 国生物工程杂志]     

PS1/GFP融合蛋白对PS1的亚细胞定位与功能的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系。本文构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即本实验条件下,PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上。     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响。应用基因芯片技术对于pcDNA3.1()和pcDNA3.1()PS1TP1分别转染的HepG2细胞的基因表达谱进行分析。以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1()PS1TP1。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析并比较。在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调。PS1TP1基因的表达对于肝细胞基因表达谱有显著影响。DNA芯片技术是分析反式调节靶基因的有效技术途径。     

FAM92A1-289基因的真核表达载体构建和亚细胞定位

利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。经双酶切和核酸序列分析证实重组质粒包含有正确编码的FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。     

Detergent-dependent dissociation of active gamma-secretase reveals an interaction between Pen-2 and PS1-NTF and offers a model for subunit organization within the complex

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.     

Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries

Low-temperature (77 K) fluorescence emission spectra of intact cells of a cyanobacterium, Synechocystis sp. PCC 6714, and a green alga, Chlamydomonas reinhardtii, were quantitatively analyzed to examine differences in PS I/PS II stoichiometries. Cells cultured under different spectral conditions had various PS I/PS II molar ratios when estimated by oxidation-reduction difference absorption spectra of P700 (for PS I) and Cyt b-559 (for PS II) with thylakoid membranes. The fluorescence emission spectra under the Chl a excitation at 435 nm were resolved into several component bands using curve-fitting methods and the relative band area between PS II (F685 and F695) and PS I (F710 or F720) emissions was compared with the PS I/PS II stoichiometries of the various cell types. The results indicated that the PS I/PS II fluorescence ratios correlated closely with photosystem stoichiometries both in Synechocystis sp. PCC 6714 and in C. reinhardtii grown under different light regimes. Furthermore, the correlation between the PS I/PS II fluorescence ratios and the photosystem stoichiometries is also applicable to vascular plants.     

B19病毒XA株VP1独特区真核表达系统的建立及鉴定

目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体.方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体plRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体plRES2-EGFP-VP1u.将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达.结果:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达.结论:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础.     

Effect of P-chirality of internucleotide bonds on B-Z conversion of stereodefined self-complementary phosphorothioate oligonucleotides of the [PS]-d(CG)4 and [PS]-d(GC)4 series

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.     

GII型诺如病毒VP2蛋白在昆虫细胞中的表达与定位研究

【目的】利用杆状病毒表达系统表达诺如病毒(GenegroupⅡ)VP2蛋白,分析其亚细胞定位,为深入研究VP2蛋白的功能奠定基础。【方法】设计可扩增完整ORF3基因片段的引物P1和P2,在下游引物中引入6×His标签的编码序列,从质粒pMD-ORF3中克隆了含有6×His编码序列的ORF3基因,与pFastBac1载体连接,构建重组质粒pFB-ORF3,转化DH10Bac感受态细胞获得重组杆状病毒基因组Bac-ORF3,脂质体介导转染sf9昆虫细胞获得表达VP2蛋白的重组杆状病毒Ac-VP2,感染sf9细胞后,收集病变细胞,采用抗6×His标签的单克隆抗体作为一抗进行Western blot与间接免疫荧光实验鉴定。【结果】Western blot实验证实Ac-VP2感染的sf9细胞在约29 kD处出现特异性条带;间接免疫荧光实验证实Ac-VP2感染的sf9细胞出现特异性绿色荧光,并且VP2主要定位于sf9的细胞核与细胞膜。【结论】诺如病毒VP2蛋白在Ac-VP2感染的sf9细胞中获得成功表达,并且主要定位于sf9细胞的细胞核与细胞膜。     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

Phosphorylation of PS II polypeptides inhibits D1 protein-degradation and increases PS II stability

To study the significance of Photosystem (PS) II phosphorylation for the turnover of the D1 protein, phosphorylation was compared with the synthesis and content of the D1 protein in intact chloroplasts. As shown by radioactive labelling with [³²P_i] phosphorylation of PS II polypeptides was saturated at light intensities of 125 mol m^-2 s^-1. Under steady state conditions, in intact chloroplasts D1 protein, once it was phosphorylated, was neither dephosphorylated nor degraded in the light. D1 protein-synthesis was measured as incorporation of [¹⁴C] leucine. As shown by non-denaturing gel-electrophoresis followed by SDS-PAGE newly synthesised D1 protein was assembled to intact PS II-centres and no free D1 protein could be detected. D1 protein-synthesis was saturated at light intensities of 500 mol m^-2 s^-1. The content of D1 protein stayed stable even after illumination with 5000 mol m^-2 s^-1 showing that D1 protein-degradation was saturated at the same light intensities. The difference in the light saturation points of phosphorylation and of D1 protein-turnover indicates a complex regulation of D1 protein-turnover by phosphorylation. Separation of the phosphorylated and dephosphorylated D1 protein by LiDS-gelelectrophoresis combined with radioactive pulse-labelling with [¹⁴C] leucine and [³²P_i] revealed that D1 protein, synthesised under steady state conditions in the light, did not become phosphorylated but instead was rapidly degraded whereas the phosphorylated form of the D1 protein was not a good substrate for degradation. According to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 protein-turnover. Fractionation of thylakoid membranes confirms that the phosphorylated, non-turning over pool of PS II-centres was located in the central regions of the grana, whereas PS II-centres involved in D1 protein-turnover were found exclusively in the stroma-lamellae and in the grana-margins.Abbreviations chl chlorophyll - F_v yield of variable fluorescence, difference between F_m, the maximal fluorescence yield at saturating light, when all reaction-centres are closed, and F_o, the fluorescence yield in the dark, when all reaction-centres are open - LHC light harvesting complex - PFD photon flux density - PS photosystem     

Regulation of amyloid precursor protein processing by presenilin 1 (PS1) and PS2 in PS1 knockout cells

The presenilin 1 (PS1) and PS2 proteins are thought to play roles in processing of amyloid precursor protein (APP), but the nature of this role is not fully understood. Recent studies have shown that PS1 is necessary for cleavage of APP at the gamma-secretase site. We now show that PS1 and PS2 participate in other aspects of APP processing. Fibroblasts generated from PS1 knockout mice have increased levels of the APP cleavage products, secreted APP (APPs), and APP C-terminal fragments, but lower secretion of APPs and Abeta. We have also observed that loss of PS1 prevents protein kinase C or extracellular regulated kinase from increasing production of the APP cleavage products, APPs, and APP C-terminal fragments. Transfection of PS1 -/- cells with PS1 restores the responsiveness of APP processing to protein kinase C and extracellular regulated kinase. This suggests that the changes in APP processing in PS1 -/- cells result strictly from the absence of PS1. Transfection of PS1 -/- cells with PS2 is also able to correct the deficits in APP secretion, which suggests that the PS2 also has the ability to regulate APP processing. Finally, transfection of the truncated PS2 construct, Alg3, into cells lacking PS1 increases APP C-terminal fragments. This suggests that Alg3 can interfere with the processing of APP by PS2. These data point to roles for both PS1 and PS2 in regulating APP processing and suggest that the role of these proteins also includes coupling APP to signal transduction pathways.     

Transport of exogenous fluorescent phosphatidylserine analogue to the Golgi apparatus in cultured fibroblasts

We have examined intracellular transport and metabolism of the fluorescent analogue of phosphatidylserine, 1-palmitoyl-2-(N-[12[(7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino] dodecanoyl])-phosphatidylserine ([palmitoyl-C12-NBD]-PS) in cultured fibroblasts. When monolayer cultures were incubated with liposomes containing (palmitoyl-C12-NBD)-PS at 37 degrees C, fluorescent PS was transported to the Golgi apparatus. NBD-containing analogues of phosphatidylcholine, phosphatidylethanolamine (PE), or phosphatidic acid did not accumulate in the Golgi apparatus under the same experimental conditions. We suggest that the transport is not due to endocytosis, but is the result of incorporation and trans-bilayer movement of the (palmitoyl-C12-NBD)-PS at the plasma membrane followed by translocation of the lipid from plasma membrane to the Golgi apparatus via nonvesicular mechanisms. Uptake of fluorescent PS was inhibited by depletion of cellular ATP and was blocked by structural analogues of the lipid or by pretreatment of cells with glutaraldehyde or N-ethylmaleimide. After incorporation into the cell, fluorescent PS was metabolized to fluorescent PE. The intracellular distribution of fluorescence changed during the conversion. In addition to the Golgi apparatus, mitochondria also became labeled.     

The O18 antigens (lipopolysaccharides) of Escherichia coli. Structural characterization of the O18A, O18A1, O18B and O18B1-specific polysaccharides.

The O-specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L-rhamnose (Rha), N-acetyl-D-glucosamine, D-galactose, and D-glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one- and two-dimensional NMR spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3. In all four polysaccharides alpha-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is beta-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is alpha-D-Glcp-(1 in O18B-PS and O18B1-PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1-PS and O18B1-PS the alpha-D-GlcpNAc residue A is substituted with alpha-Glcp-(1 (residue F), which is linked to O-6 in O18A1-PS and to O-4 in O18B1-PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.     

HPV16L1核浆运输的动力学过程

利用增强型绿色荧光蛋白(Enhancegreenflurenscentprotein,EGFP)标记不同的截短型HPV16L1蛋白(Humanpapillomavirustype16L1protein,HPV16L1),分析HPV16L1蛋白核定位信号(Nucleuslocationsignal,NLS)的作用。构建重组pFB-EGFP、pFB-EGFP-HPV16L1、pFB-EGFP-HPV16L1△NLS和pFB-EGFP-NLSHPV16L1p转移载体;在DH10Bac宿主菌内经Tn7转座子介导的同源重组后转染Sf-9细胞,获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS和Ac-EGFP-NLSHPV16L1杆状病毒,感染Sf-9昆虫细胞表达相应截短型HPV16L1融合蛋白;利用荧光显微镜和激光共聚焦显微镜观察不同融合蛋白的荧光特性和核浆转运动力学过程。结果发现Ac-EGFP杆状病毒感染的Sf-9细胞内明亮的绿色荧光均匀分布;重组Ac-EGFP-HPV16L1和Ac-EGFP-NLSHPV16L1杆状病毒感染的Sf-9细胞,明亮的绿色荧光主要位于细胞核内;重组Ac-EGFP-HPV16L1△NLS杆状病毒感染的Sf-9细胞,绿色荧光局限于细胞浆内,细胞核内无绿色荧光。说明HPV16L1蛋白羧基端的23个氨基酸(GKRKATPTTSSTSTTAKRKKRKL)具有完全核定位作用,能引导HPV16L1蛋白和EGFP突破核膜屏障进入Sf-9细胞核内。     

一种包装AAV2/5的重组单纯疱疹病毒的构建

为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。     

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.     

利用反式剪接核酶修复突变绿色荧光蛋白mRNA

为构建修复突变绿色荧七蛋白（GFP）基因的反式剪接核酶,分别构建包含突变的GFP基因的XYQ5／10-pGEM重组质粒、XYQ5／10—pEGFP—C2重组质粒及用于修复该突变基因的反式剪接核酶载体trans—rib—CMV2。通过对体外共转录XYQ5／10—pGEM和trans—rib—CMV2重组质粒的RNA产物进行RT—PCR检测核酶细胞外剪接效果;通过XYQ5／10-pEGFP-C2和trans—rib—CMV2重组质粒共转染HeLa细胞检测核酶细胞内的剪接效果。结果显示,XYQ5／10—pGEM、XYQ5／10-pEGFP-C2及trans—rib—CMV2重组质粒构建成功,反式剪接核酶在细胞外及细胞内都可以修复突变基因。虽然效率不高,但为今后更大规模地研究设计反式剪接核酶打下了基础。