植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,牵涉着大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本文利用病毒SV40抗原蛋白中的核定位信号（nuclear localization signal,NLS）标记GFP蛋白,通过拟南芥细胞质的介导,利用HeLa细胞核建立起了研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在HeLa细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过HeLa细胞建立起的半细胞体系能为蛋白入核转运研究提供一个有效的研究体系。     

A Protein Nuclear Extract from D. melanogaster Larval Tissues

Preparation of protein nuclear extracts is often the first step to study in vitro biological processes occurring in the nucleus of the eukaryotic cell. Nuclear extracts have been extensively used in different model organisms to identify and study protein function in nuclei. Drosophila embryos can be collected in large quantities and have been the source of choice for the production of protein nuclear extracts. However, most of Drosophila in vivo studies on protein function are conducted in larval tissues. Here we report a new method to produce highly stable large-scale protein nuclear extracts from whole Drosophila larvae that are suited for a variety of biochemical analyses.     

Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR-binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super-resolution methods, single-molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR-mediated sites. We found that the direct binding sites had a maximum at approximately −30 nm with regard to the NPC center, whereas the indirect transport-relevant binding sites peaked at approximately −10 nm. The 20 nm-shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single-molecule microscopy. Then we analyzed the distribution of the NTR Kapβ1 and a Kapβ1-based transport complex and found them to have also binding maxima at approximately −10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.     

细胞提取物重编程研究进展

体细胞重编程是在特定的条件下使已分化的细胞转变成为另一种细胞.体细胞重编程的方式主要有体细胞核移植技术、细胞融合技术、细胞提取物处理技术及特定转录因子转染技术.现有研究表明,细胞提取物重编程技术在体细胞重编程中发挥着一定的作用,为此,就该技术的最新研究进展和可能机制作一综述.     

Nuclear antigens in the HeLa cell cycle

Summary Antisera to 0.35 M NaCl extracts and residues of S phase HeLa nuclei were reacted with electrophoretically separated proteins from the nuclei or nuclear material of HeLa cells synchronized in G₁, S, G₂ or M phases of the cell cycle. Quantitative evaluation of the peroxidase-antiperoxidase stained nitrocellulose transfers (Western blots) revealed significant changes in the quantities of nuclear non-histone proteins during the cell cycle. Immunochemical staining of electrophoretically separated nuclear antigens permits their selective detection in minute quantities and in the presence of many additional proteins.     

龙须菜叶绿素- 蛋白复合物的分离及鉴定

以海洋经济海藻——龙须菜（Gracilaria lemaneiformis）为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物。P700差示光谱鉴定分离的光系统Ⅰ（PSⅠ）颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ（PSⅡ）颗粒。结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒。     

Influence of Selenium on Mast Cell Mediator Release

Selenium supplementation still enhanced the immune response even in individuals who, according to current standards, would be considered as not being overtly selenium deficient. Mast cells are granulated cells that play a pivotal role in allergic reactions. In this study, we investigated the modulatory effect of sodium selenite on mediator release and degranulation of murine mast cell line (MC/9). Cells were pre-treated with selenium selenite (1, 2, 3 μg/ml) for 24 h and controls left untreated. Then, cells were sensitized overnight with anti-dinitrophenyl (DNP) IgE and challenged with DNP/HSA for degranulation induction. The histamine and prostaglandin D2 (PGD2) were measured by ELISA, and β-hexosaminidase was measured by spectrophotometery method. Selenium-treated cells revealed significant decrease in concentration of PGD2 (P?=?0.019) and β-hexosaminidase (P?=?0.009). In addition, a slight reduction of histamine release by the selenium-treated cells was observed, based on our intracellular and extracellular assessments. The most inhibitory effect of selenium supplementation on mediator release of MC/9 cells was obtained in the presence of 3 μg/ml of sodium selenite. The results of the present study demonstrate beneficial effects of supplemental selenium in attenuating clinical manifestations of allergy and asthma.     

Interaction of Human Ribosomal Protein S26 with Fragments of Its Own mRNA and Pre-mRNA in Nuclear Extract of HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (K _a) 5.0 · 10⁷ M^–1) and, to a lesser extent, to the rpS26 mRNA (K _a 2.0 · 10⁷ M^–1). The binding was specific, since human rpS19 had an order of magnitude lower K _a with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48K, 59K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

龙须菜叶绿素-蛋白复合物的分离及鉴定

以海洋经济海藻--龙须菜(Gracilarialemaneiformis)为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物.P700差示光谱鉴定分离的光系统Ⅰ(PSⅠ)颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ(PSⅡ)颗粒.结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒.     

Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei.

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.     

Characterization of lamina-bound chromatin in the nuclear shell isolated from HeLa cells

The structure and the polypeptide composition of the nuclear shell isolated from interphase HeLa cells have been investigated and compared to those of the intranuclear material. The isolated nuclear shell contains chromatin superstructures (28-32 nm thick fibres) made of tightly packed nucleosomes that resist low ionic strength conditions and that are associated with the three nuclear lamins. Chromatin in the nuclear shell exhibits very simple chemical composition. Especially, non-histone proteins are lacking. The results presented here rule out the possibility that the nuclear shell results from contamination of lamina by intranuclear elements. They suggest that the lamins are directly involved in the specific properties and in the organization of chromatin in the nuclear shell.     

Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).     

Structure-Based Characterization of Multiprotein Complexes

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Functional Characterization of Septin Complexes

Septins belong to a family of conserved GTP-binding proteins found in majority of eukaryotic species except for higher plants. Septins form nonpolar complexes that further polymerize into filaments and associate with cell membranes, thus comprising newly acknowledged cytoskeletal system. Septins participate in a variety of cell processes and contribute to various pathophysiological states, including tumorigenesis and neurodegeneration. Here, we review the structural and functional properties of septins and the regulation of their dynamics with special emphasis on the role of septin filaments as a cytoskeletal system and its interaction with actin and microtubule cytoskeletons. We also discuss how septins compartmentalize the cell by forming local protein-anchoring scaffolds and by providing barriers for the lateral diffusion of the membrane proteins.     

综述: 细胞提取物重编程研究进展

体细胞重编程是在特定的条件下使已分化的细胞转变成为另一种细胞.体细胞重编程的方式主要有体细胞核移植技术、细胞融合技术、细胞提取物处理技术及特定转录因子转染技术.现有研究表明,细胞提取物重编程技术在体细胞重编程中发挥着一定的作用,为此,就该技术的最新研究进展和可能机制作一综述.     

Identifying Heteroprotein Complexes in the Nuclear Envelope

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.     

Influence of Optical Properties of Ag NPs from Raphanus sativus Leaf Extract on Lanthanide Complexes

Plasmonics - Simple rapid green synthesis route using Raphanus sativus leaf extract as reducing and stabilizing agent produced silver nanoparticles (Ag NPs) at room temperature. The formation of Ag...